Journal: Leukemia

Article Title: Spred1 deficit promotes treatment resistance and transformation of chronic phase CML

doi: 10.1038/s41375-021-01423-x

Figure Lengend Snippet: A Expression of SPRED1 in BM CD34+ cells from patients with BC CML and CP CML by Q-RT-PCR (n=8 samples for BC CML and n=12 samples for CP CML) and western blot and in BM by immunohistochemistry staining (one of three independent experiments with similar results was shown) (left), and expression of miR-126 in CD34+ and CD34+CD38− cells from BC CML (n=6 samples) and CP CML (n=10 samples) patients by Q-RT-PCR (right). B SPRED1 mRNA expression by Q-RT-PCR and protein expression by western blot, miR-126 levels by Q-RT-PCR, cell cycling by Ki-67 and DAPi staining (top) or by cell trace violet staining (bottom) followed by flow cytometry analysis in CML CD34+ cells transduced with SPRED1 siRNA to knock-down (KD) SPRED1 or with a non-targeting control siRNA (Ctrl). UND: undivided cells, G0; DIV: division. C Representative colonies and quantification of colony forming cells (CFC) in CML CD34+ (left) and CD34+CD38− (right) cells transduced with Spred1 siRNA to KD SPRED1 or with ctrl siRNA (n=3). Results shown represent mean ± SEM. Significance values: *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Human CD34 + cells were selected using the indirect CD34 microbead kit (Miltenyi Biotec, San Diego, CA) and CD34 + CD38 − cells were sorted after staining with human antibodies against CD34 and CD38 ( Supplementary Table 1 ) or selected using CD34+CD38- cell isolation kit (Miltenyi Biotec, San Diego, CA) according to the manufacturer’s protocol.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining, Flow Cytometry, Transduction, Knockdown, Control

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Multi-omics analysis reveals potential regulatory roles of SPI1 in EHT (A) Heatmap of 575 expressed TFs during the differentiation of Pluripotent Stem Cells (PSC) into Hematopoietic Progenitor Cells (HPC). Four expression clusters (TC1-TC4) are identified and are denoted by color and number. (B) Line graph of variable accessible chromatin elements during PSC to HPC differentiation, again with four identified clusters (C1-C4). (C) Enriched TF motifs in peaks of clusters C1-C4. (D) Heatmap showing variations in TF activity among the top 50 active transcription factors during differentiation. (E) Pearson correlation analysis of TF activity and gene expression for five key TFs. (F) Visualization of ATAC-seq footprint for motifs of the five representative TFs across different developmental stages. See also Figure S2 and .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Biomarker Discovery, Expressing, Activity Assay, Gene Expression

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Knockdown of SPI1 at the in vitro -generated HECs stage severely impairs HPC generation (A) Schematic illustration of the in vitro differentiation approach. SPI1 siRNA or control siRNA were introduced on D4 of EHT, with evaluations from D5 to D8. (B) Flow cytometric sorting strategy for HECs treated with siRNA. (C) qPCR results showing SPI1 expression from D5 to D8, using two distinct siRNA sequences targeting SPI1 . (D) Western blot analysis of PU.1 expression in siCtrl and siSPI1_1/2 groups from D5 to D8, with GAPDH as the internal normalization control. (E) Representative flow cytometry density plots showing CD34 + CD43 + cells among total cells in siCtrl and siSPI1_1/2 groups, from D5 to D8. (F) Quantification of suspended CD34 + CD43 + HPCs from siCtrl and siSPI1_1/2 groups, from D5 to D8. (G) Microscopy images of cells on D8 for siCtrl and siSPI1 conditions, scale bar = 100 μm. (H) Representative flow cytometry density plots of CD34 + CD43 + HPCs in suspended cells for siCtrl and siSPI1_1/2 groups on D8. Data are from n = 3 independent experiments, and statistical significance was determined using two-tailed unpaired Student’s t-test. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Figure S4 , Table S1 and Table S2 .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Knockdown, In Vitro, Generated, Control, Expressing, Western Blot, Flow Cytometry, Microscopy, Two Tailed Test

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Knockdown of SPI1 during EHT does not alter HPC proliferation or apoptosis but affects differentiation potential (A) Flow cytometry sorting strategy for D8 suspended CD34 + CD43 + HPCs (FAM +/− ). (B and C) Representative flow cytometry density plots (B) and frequency plot (C) for cell cycle, G0/G1 (Edu − Hoechst low ), S (Edu + Hoechst low/high ) and G2/M (Edu − Hoechst high ) on D8 suspended CD34 + CD43 + HPCs (FAM +/− ) in siCtrl and siSPI1_1/2 groups. (D and E) Representative flow cytometry density plots (D) and frequency plot (E) of early apoptosis (Annexin V + DAPI − ) and late apoptosis (Annexin V + DAPI + ) on D8 suspended CD34 + CD43 + HPCs (FAM +/− ) in siCtrl and siSPI1_1/2 groups. Significance determined by two-tailed unpaired Student’s t-test. (F) Colony Forming Unit (CFU) assay of FAM − CD34 + CD43 + HPCs generated on D8 under siCtrl and siSPI1_1/2 conditions. Unless otherwise stated, data are from n = 3 independent experiments, and statistical significance was determined using two-way ANOVA. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance. See also Figure S5 .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Knockdown, Flow Cytometry, Two Tailed Test, Colony-forming Unit Assay, Generated

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Transcriptional regulatory network suggests LYL1 and KLF1 as potential downstream targets of SPI1 (A) Heatmap displaying reordered normalized trans -regulation score (TRS) at the HPC stage. Identified modules from nonnegative matrix factorization (NMF) are represented in different colors. (B) Boxplots showing gene expression levels in different modules from A, comparing Endothelial Progenitor Cells (EPC) and HPC. p values, two-tailed paired Student’s t test. (C) All SPI1 Target Genes (TGs) are ordered descendingly according to their TRS, with a focus on the top 10 transcription factors (TFs) within these TGs. (D) Violin plots showing expression levels of the top 10 TFs from SPI1 TGs in both in vivo AGM hematopoiesis (left) and the in vitro system (right). (E) qPCR results showing the expression of the top 10 transcription factors from SPI1 target genes in D8 CD34 + CD43 + HPCs, excluding non-expressed genes SNAI3 and RUNX3 , in siCtrl and siSPI1_1/2 groups. Data are from n = 3 independent experiments, with significance assessed using two-tailed unpaired Student’s t test. (F) Enrichment analysis of Gene Ontology (GO) terms and KEGG signaling pathways of SPI1 ’s TGs that have p -value <0.001. Data are presented as means ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance.

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Gene Expression, Two Tailed Test, Expressing, In Vivo, In Vitro, Protein-Protein interactions

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Overexpression of LYL1 and KLF1 can rescue SPI1 knockdown phenotypes (A) Co-transfection strategy of SPI1 siRNA with KLF1 / LYL1 overexpression during EHT to assess D8 HPC differentiation potential. (B) Western blot analysis of KLF1/LYL1 from D6 differentiated cells in the siCtrl+OE-Ctrl, siSPI1, and siSPI1+OE-KLF1/LYL1 groups, normalized to GAPDH. (C and D) Flow cytometry analysis of suspended CD34 + CD43 + HPCs on D8: (C) Representative density plot and (D) quantification in siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups. n = 3 independent experiments. (E) Colony-forming units (CFU) assay on D8 suspended CD34 + CD43 + HPCs across siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups, with significance assessed using two-way ANOVA. (F) Schematic description of lymphoid differentiation. (G) Representative flow cytometry density plots of the distribution of CD45 -/mid/+ CD5 + CD7 + cells under the normal conditions at D29. (H–I) Analysis of CD45 + CD5 + CD7 + cells: (H) Representative flow cytometry density plots (H) and frequency plot (I) of CD45 + CD5 + CD7 + cells in siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups. Unless otherwise stated, data are from n = 3 independent experiments, and statistical significance was determined using two-tailed unpaired Student’s t-test. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance. See also Figures S6 and .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Over Expression, Knockdown, Cotransfection, Western Blot, Flow Cytometry, Colony-forming Unit Assay, Two Tailed Test

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet:

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Recombinant, Transfection, Plasmid Preparation, Reverse Transcription, In Vivo, In Vitro, Biomarker Discovery, Software

Journal: bioRxiv

Article Title: Effects of Post-Myocardial Infarction Heart Failure on the Bone Vascular Niche

doi: 10.1101/2020.05.29.123711

Figure Lengend Snippet: ( a, b ) Flow cytometry analysis of healthy and HF patient bone marrow. ( a ) Gating strategy for endothelial cells. Representative flow cytometry dot plots showing EC subsets with distinct expression of CD31 and Endomucin (EMCN) in healthy and HF bone marrow aspirates (gated on CD45 neg Lin neg viable single cells). ( b ) Type H endothelial cells are reduced in the HF patients compared to healthy controls (left panel), while the total number of endothelial cells remains unchanged (right panel) (N=8 for healthy, N=18 for HF patients). Data are shown as mean ± SEM. P-value was calculated by unpaired, Mann Whitney test. ( c - i ) scRNA-seq of a post-MI heart failure and an aged-matched healthy control. ( c , d ) Clustered cells from both subjects are displayed in t-SNE plots, colored by cluster (left), cell annotation (middle) and health status (right). ( d ) Expression of EMCN and PECAM1. EMCN is enriched in the cells corresponding to cluster 0. ( e - f ) Analysis of EMCN enriched cell cluster 0 population. ( e ) Dichotomization in EMCN enriched population shown in t-SNE plot (Left). Relative expression of key genes in the EMCN enriched population represented by features plots as indicated. ( f ) Violin plots showing the relative expression of key genes in the EMCN enriched population, confirming the significantly increased expression of IL1B and MYC in the HF patient. ( g ) Distribution of cells along pseudotime trajectory branchpoints. Pseudotime analysis revealed 13 states. ( h ) Distribution of cells among pseudotime states and relative IL1B expression. Distribution analysis revealed that states 10, 11, 12, and 13 are mainly populated by HF patient cells. IL1B expression is higher in states 12 and 13. Dashed line indicates normalized Unique Molecular Identifier (nUMI) counts of 2.5. ( i ). Gene Ontology term ranking of upregulated genes in pseudotime state 13.

Article Snippet: Lineage negative and CD31 positive BMCs were isolated using first immunomagnetic Lineage Cell Depletion Kit (130-092-211, Miltenyi Biotec) followed by positive selection with CD31 MicroBead Kit (130-091-935, Miltenyi Biotec) using a magnetic cell separation device (QuadroMACS Separator;130-090-976, Miltenyi Biotec).

Techniques: Flow Cytometry, Expressing, MANN-WHITNEY, Control

Journal: bioRxiv

Article Title: Tim-3 co-stimulation promotes short-term effector T cells, restricts memory precursors and is dispensable for T cell exhaustion

doi: 10.1101/179002

Figure Lengend Snippet: A , CD25-depleted T cells from Nur77 GFP mice were stimulated for three days with anti-CD3/CD28 mAbs, followed by a seven day rest with IL-2. After three rounds of stimulation, cells were stained for PD-1 and Tim-3 and analyzed by flow cytometry. Representative of 5 technical replicates per experiment, repeated with n=5 mice. B , WT C57BL/6 mice were infected with LCMV Arm. After 30d, spleens were harvested for flow cytometry (n=5 mice, mean ± SD). representative of three independent experiments C , Tim-3 + vs. Tim-3 − CD8 + cells were further analyzed for expression of activation and differentiation markers shown in the histograms. D , splenocytes from the same experiments as panels b-c were stained with LCMV tetramers plus α Tim-3. (n=5 mice, mean ± SD) representative of three independent experiments. **p<0.01 by two-tailed paired Student’s t test. E , C57Bl/6 mice previously infected with LCMV-Arm (>30d.p.i.) were challenged with LM-GP33 and Tet − vs. Tet + CD8 + splenocytes were analyzed for KLRG1 and Tim-3 four days post-challenge. Representative of three independent experiments (n=5).

Article Snippet: CD25 depletion was done using a CD25 microbead kit (Miltenyi).

Techniques: Staining, Flow Cytometry, Infection, Expressing, Activation Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Plasmid-based donor templates enable efficient nonviral gene editing of TRAC locus in primary T cells. (A–C) Titration of linear dsDNA donor template. (A) Diagram of linear dsDNA knock-in construct TRAC -mNG. (B) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) 3 d after electroporation with 1, 2, 4, 6, or 8 µg of linear dsDNA donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). (C) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. (D–F) Titration of pUC57 plasmid donor template. (D) Diagram of pUC57 knock-in construct TRAC -mNG. (E) Bar graphs showing the frequency of CD8 + T cells expressing mNG, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) 3 d after electroporation with 1, 2, 4, 6, or 8 µg of pUC57 plasmid donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). (F) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. (G–I) Titration of nanoplasmid donor template. (G) Diagram of nanoplasmid knock-in construct TRAC -mNG. (H) Bar graphs showing the frequency of CD8 + T cells expressing mNG, total cell recovery, and edited cell recovery (mNG-positive cells) 3 d after electroporation with 1, 2, 4, 6, or 8 µg of nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). (I) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. This experiment was performed twice. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 in RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Plasmid Preparation, Titration, Knock-In, Construct, Cell Recovery, Electroporation, Expressing

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Optimization of nonviral gene editing in primary T cells using plasmid-based donor templates. (A–F) Titration of linear dsDNA and nanoplasmid donor templates in CD8 + T cell cultures in RPMI/10% FBS medium. (A) Diagram of linear dsDNA knock-in construct TRAC -mNG. (B) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. (C) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) of CD8 + T cells cultured in RPMI/10% FBS 3 d after electroporation with 1, 2, or 4 µg of linear dsDNA donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (D) Diagram of nanoplasmid knock-in construct TRAC -mNG. (E) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. (F) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) of CD8 + T cells cultured in RPMI/10% FBS 3 d after electroporation with 1, 2, or 4 µg of nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (G) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery 3 d after electroporation with 2 µg of either linear dsDNA or nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus in the presence of absence of PGA. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed twice. (H) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery 3 d after electroporation with 2 µg of either linear dsDNA or nanoplasmid donor template that either did or did not contain truncated Cas9 target sequences (tCTS) together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment has been performed twice. *, P < 0.05; **, P < 0.01; ***, P < 0.001 in RM one-way ANOVA with Geisser–Greenhouse correction (C and F) or paired t test (G and H).

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Plasmid Preparation, Titration, Knock-In, Construct, Expressing, Cell Recovery, Cell Culture, Electroporation

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Cytokine production and stress response induced in T cells following exposure to dsDNA donor templates. (A) IFN-α measured by Simoa and IFN-γ, TNF-α, and IL-2 measured by Luminex from CD8 + T cells 18 h after transfection with Cas9-RNP targeting the TRAC locus alone or together with nanoplasmid donor template compared with non-transfected control T cells (No RNP). Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed once for Simoa and twice for Luminex. (B) GSEA from RNA-sequencing of CD8 + T cells after transfection with Cas9-RNP targeting the TRAC with nanoplasmid donor template compared with Cas9-RNP alone. Gene sets for IFN-γ response, IFN-α response, TNF-α signaling, and inflammatory response were significantly enriched. (C) GSEA from RNA-seq of CD8 + T cells after transfection with Cas9-RNP targeting the TRAC with linear dsDNA donor template compared to Cas9-RNP alone. Gene sets for IFN-γ response, IFN-α response, TNF-α signaling, and inflammatory response were significantly enriched. (B and C) The y axis represents enrichment score, and on the x axis are genes (vertical black lines) represented in gene sets. The colored band at the bottom represents the degree of differentially expressed genes (red for upregulation and blue for downregulation). (D) Gene set enrichment analysis of all 375 upregulated genes in both Nanoplasmid/Cas9-RNP and linear dsDNA/Cas9-RNP over Cas9-RNP-only using the GSEA MSigDB Hallmark 2020. (E–H) Heatmaps showing upregulated genes in Nanoplasmid/Cas9-RNP and linear dsDNA/Cas9-RNP over Cas9-RNP-only that mostly contributed to IFN-α response (E), TNF-α response (F), apoptosis (G), or inflammatory response (H; all MSigDB Hallmark). Color-coded by the normalized RNA-seq count data with variance stabilizing transformation (VST). This experiment was performed once. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 in one-way ANOVA.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Luminex, Transfection, Control, RNA Sequencing, Transformation Assay

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Optimization of CRISPR/Cas9-mediated gene knock-in with plasmid-based donor DNA in CD4 + and CD8 + T cells. (A and B) Homology arm optimization for plasmid-based donor templates. (A) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. (B) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) 3 d after electroporation with pUC57 plasmid or nanoplasmid donor templates with homology arm lengths between 100 bp and 2,000 bp (amounts equimolar to 4 µg of the pUC57 2,000 bp construct) together with Cas9-RNP targeting the TRAC locus ( n = 2). Circles represent individual donors; bars represent median values with range. This experiment was performed three times. (C) Frequency of CD8 + T cells expressing mNG, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) 3 d after electroporation after stimulating cells for 24, 36, 48, or 72 h prior to electroporation with nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus ( n = 4). Circles represent individual donors; bars represent median values with range. This experiment was performed twice. (D) Nucleofection pulse code optimization in CD8 + T cells electroporated with nanoplasmid donor template and Cas9-RNP targeting the TRAC locus. Graph shows frequency of cells expressing mNG and edited cell recovery (mNG-positive cells) 3 d after electroporation. Each circle represents a distinct pulse code. Data are representative of three independent CD8 + T cell donors. This experiment was performed twice. (E and F) Gene editing targeting the TRAC locus in CD4 + T cells. Representative contour plot showing the frequency of CD4 + T cells expressing mNG (E) and bar graphs (F) depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) 5 d after electroporation of CD4 + T cells with TRAC -mNG nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus ( n = 3). Circles represent individual donors; bars represent median values with range. This experiment was performed twice. *, P < 0.05; **, P < 0.01 in RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: CRISPR, Gene Knock-In, Plasmid Preparation, Expressing, Knock-In, Cell Recovery, Electroporation, Construct

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Nonviral TCR editing using plasmid DNA donors. (A) Diagram of TCR α and β genomic loci. V gene (purple), D gene (red), J gene (blue), and constant region (green) segments. sg TRAC and sg TRBC targeting sites are indicated. (B) Diagrams of nanoplasmid knock-in constructs TRAC -1G4TCR, TRAC -TCR6-2, and TRAC -CD19CAR. (C, E, and G) Representative contour plots (left) and bar graphs (right) showing the frequencies of CD8 + T cells expressing (C) a NY-ESO-1-specific 1G4 TCR, (E) a CMV-specific pp65 6-2 TCR, and (G) a CD19-CAR 5 d after electroporation using nanoplasmid donor templates together with Cas9-RNPs targeting the TRAC locus. (D, F, and H) Bar graphs showing the cell viability, total cell recovery, and edited cell recovery 5 d after electroporation using nanoplasmid donor templates encoding (D) a NY-ESO-1–specific 1G4 TCR, (F) a CMV-specific pp65 6-2 TCR, and (H) a CD19-CAR together with Cas9-RNPs targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed three times. (I) Lactate levels in culture supernatant analyzed by luminescence using the Lactate-Glo Assay were measured 1, 3, 5, and 7 d after transfection of CD8 + T cells with sg TRAC /sg TRBC Cas9-RNP (RNP only) or sg TRAC /sg TRBC Cas9-RNP and nanoplasmid donor template targeting the TRAC locus (RNP + nanoplasmid) compared with non-transfected control T cells (No RNP); RLU, relative light units. (J) Number of cells recovered from cultures 7 d after transfection of CD8 + T cells with sg TRAC /sg TRBC Cas9-RNP (RNP only) or sg TRAC /sg TRBC Cas9-RNP and nanoplasmid donor template targeting the TRAC locus (RNP + nanoplasmid) compared with non-transfected control T cells (No RNP). This experiment was performed three times. *, P < 0.05 in RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Plasmid Preparation, Knock-In, Construct, Expressing, Electroporation, Cell Recovery, Glo Assay, Transfection, Control

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Nonviral TCR editing in CD4 + and CD8 + T cells using plasmid DNA donors. (A) TCR expression on the cell surface by flow cytometry of CD8 + T cells 48 h after transfection with Cas9-RNP targeting the TRAC (sg TRAC ) or TRBC (sg TRBC ) loci. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed three times. (B–G) TCR editing in CD4 + T cells. Representative contour plots showing the frequencies of CD4 + T cells expressing a NY-ESO-1-specific 1G4 TCR (B), a CMV-specific pp65 6-2 TCR (D), and a CD19-CAR (F) and bar graphs showing the knock-in efficiency and cell viability 5 d after electroporation using nanoplasmid donor templates encoding a NY-ESO-1-specific 1G4 TCR (C), a CMV-specific pp65 6-2 TCR (E), and a CD19-CAR (G) together with Cas9-RNPs targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (H and I) Diagram depicting all possible translocation events between the TRAC , TRBC1 , and TRBC2 genomic loci (H). Bar graph (I) showing the frequencies of individual translocation events between the TRAC , TRBC1 , and TRBC2 genomic loci quantified by ddPCR in CD8 + T cells co-transfected with Cas9-RNPs targeting the TRAC and TRBC loci or in non-transfected control T cells. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (J and K) Representative histograms (J) and bar graphs (K) showing proportions of CD137-expressing pp65 TCR knock-in CD8 + T cells stimulated with indicated concentrations of pp65 495–503 peptide. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed twice. (L) Bar graphs showing IFN-γ and TNF-α production by pp65 TCR knock-in CD8 + T cells stimulated with indicated concentrations of pp65 495–503 peptide. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed twice. (M) Representative histograms showing the frequencies of CFSE-positive target cells and CFSE-negative reference cells in co-cultures with pp65 TCR knock-in CD8 + T cells in the absence or presence of the cognate peptide. (N) Graphs showing specific lysis calculated in the absence of peptide or with 0.1 µM of pp65 495–503 peptide. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed twice. (O) Bar graphs showing IFN-γ and TNF-α production by TCR6-2 (irrelevant TCR) or CD19-CAR knock-in CD4 + T cells from two donors (D1 and D2) in co-cultures with CD19-expressing B cells. Circles represent technical replicates; bars represent median values with range ( n = 9). This experiment was performed twice. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 in RM one-way ANOVA with Geisser–Greenhouse correction (A and K), paired t test (N), and one-way ANOVA (O).

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Plasmid Preparation, Expressing, Flow Cytometry, Transfection, Knock-In, Electroporation, Translocation Assay, Control, Lysis

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: TCR-engineered T cells recognize and kill antigen-expressing target cells. (A and B) Representative histograms (A) and bar graphs (B) showing proportion of CD137 expression of 1G4 TCR knock-in CD8 + T cells stimulated with indicated concentrations of NY-ESO-1 157–165 peptide. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (C and D) Bar graphs showing IFN-γ (C) or TNF-α (D) production by 1G4 TCR knock-in CD8 + T cells stimulated with indicated concentrations of NY-ESO-1 157–165 peptide. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (E) Representative histograms showing the frequencies of CFSE-positive target cells and CFSE-negative reference cells in co-cultures with 1G4 TCR knock-in CD8 + T cells in the absence or presence of the cognate peptide. (F) Graphs showing specific lysis calculated in the absence of peptide or with 0.1 µM of NY-ESO-1 157–165 peptide. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (G) Bar graphs showing IFN-γ, TNF-α, and granzyme B (GzmB) production by TCR knock-out or 1G4 TCR knock-in CD8 + T cells from three donors co-cultured with A-375 cells that express the NY-ESO-1 antigen. Circles represent technical replicates; bars represent median values with range ( n = 3). This experiment was performed twice. (H) Representative images for A-375 cells that express the NY-ESO-1 antigen and were labeled with a cytoplasmic dye and co-cultured with TCR knock-out CD8 + T cells (left) or 1G4 TCR knock-in CD8 + T cells (right) 2 and 18 h after culture seeding in the presence of caspase 3/7-green apoptosis reagent. Scale bars indicate 300 µm distance. (I) Representative target cell killing over time as measured by the Cas3/7-positive object count in co-cultures of A-375 cells expressing the NY-ESO-1 antigen and labeled with a cytoplasmic dye and co-cultured with TCR knock-out CD8 + T cells (open circles) or 1G4 TCR knock-in CD8 + T cells (filled circles). Mean values ± SD of six technical replicates. This experiment was performed twice with three independent donors per experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 in RM one-way ANOVA with Geisser–Greenhouse correction (B–D); paired t test (F); one-way ANOVA (G); or Tukey’s multiple comparisons test, two-way ANOVA (I).

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Expressing, Knock-In, Lysis, Knock-Out, Cell Culture, Labeling

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Kinetics of gene expression following transient transfection of linear dsDNA, plasmid, and nanoplasmid. (A) Diagram of nanoplasmid knock-in construct RAB11A -YFP. (B and C) Representative histograms showing the frequencies of CD8 + T cells expressing YFP (B) and bar graphs (C) depicting frequency of YFP expression, cell viability, total cell recovery, and edited cell recovery 3, 5, or 7 d after electroporation with promoter-containing nanoplasmid donor template together with (red) or without (blue) Cas9-RNPs targeting the RAB11A locus. Circles represent technical replicates; bars represent median values with range ( n = 3). This experiment was performed twice. (D) Diagram of linear dsDNA knock-in construct RAB11A -YFP. (E and F) Representative histograms showing the frequencies of CD8 + T cells expressing YFP (E) and bar graph (F) depicting frequency of YFP expression 3, 5, or 7 d after electroporation with promoter-containing linear dsDNA donor templates together with (red) or without (blue) Cas9-RNPs targeting the RAB11A locus. Circles represent technical replicates; bars represent median values with range ( n = 3). This experiment was performed once. (G) Diagram of pUC57 plasmid knock-in construct RAB11A -YFP. (H and I) Representative histograms showing the frequencies of CD8 + T cells expressing YFP (H) and bar graph (I) depicting frequency of YFP expression 3, 5, or 7 d after electroporation with promoter-containing pUC57 plasmid donor templates together with (red) or without (blue) Cas9-RNPs targeting the RAB11A locus. Circles represent technical replicates; bars represent median values with range ( n = 3). This experiment was performed twice. *, P < 0.05; **, P < 0.05; ***, P < 0.001 in Sidak’s multiple comparisons test with RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Gene Expression, Transfection, Plasmid Preparation, Knock-In, Construct, Expressing, Cell Recovery, Electroporation

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Generation of reporters of gene expression. (A) Diagram of nanoplasmid knock-in construct RAB11A -YFP. (B and C) Histogram overlay for YFP expression (B) and bar graphs (C) showing the frequency of YFP expression and cell viability of CD8 + T cells transfected with RAB11A -YFP nanoplasmid with or without RAB11A targeting Cas9-RNP 10 d after electroporation. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed three times. (D) Diagram of nanoplasmid knock-in construct AAVS1- mNG. (E and F) Histogram overlay for mNG expression (E) and bar graphs (F) showing the frequency of mNG expression and cell viability of CD8 + T cells transfected with AAVS1- mNG nanoplasmid with or without AAVS1 targeting Cas9-RNP 10 d after electroporation. Circles represent individual donors, and bars represent median values with range ( n = 4). This experiment was performed three times. (G) Diagram of nanoplasmid knock-in construct CD4- mNG. (H and I) Representative contour plots (H) and bar graphs (I) showing the frequency of CD4 + and CD8 + T cells expressing mNG and cell viability 10 d after electroporation of a nanoplasmid donor template and Cas9-RNP targeting the CD4 locus. Circles represent individual donors, and bars represent median values with range ( n = 4 for CD4 + T cells, n = 3 for CD8 + T cells). This experiment was performed twice. (J) Histogram overlay for CD4 expression in CD4 + T cells transfected with CD4- mNG nanoplasmid together with a non-targeting control Cas9-RNP (sgNTC) or a Cas9-RNP targeting the CD4 locus (sg CD4 ) 10 d after electroporation. (K) Diagrams of nanoplasmid knock-in constructs TNFRSF9 -mNG and RAB11A -YFP (left) and representative contour plots (right) showing the frequency of CD8 + T cells expressing CD137 and mNG after electroporation with a nanoplasmid mNG reporter construct targeting the TNFRSF9 locus or a constitutive YFP expressing construct targeting the RAB11A locus together with the respective Cas9-RNP either without restimulation or 6 h after restimulation with Transact. (L) Bar graphs showing the frequency of YFP (blue) and mNG (red) expressing CD8 + T cells over time after electroporation with a nanoplasmid mNG reporter construct targeting the TNFRSF9 locus or a constitutive YFP expressing construct targeting the RAB11A locus together with the respective Cas9-RNP and restimulation with Transact at time 0 h. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (M) Bar graphs showing the geometric mean fluorescent intensity (gMFI) of CD137 expression in CD8 + T cells over time after electroporation with a nanoplasmid mNG reporter construct targeting the TNFRSF9 locus or a constitutive YFP expressing construct targeting the RAB11A locus together with the respective Cas9-RNP and restimulation with Transact at time 0 h ( n = 4). Circles represent individual donors; bars represent median values with range. *, P < 0.05; **, P < 0.01 in paired t test (C, F, I, and J) or in RM one-way ANOVA with Geisser–Greenhouse correction (L).

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Gene Expression, Knock-In, Construct, Expressing, Transfection, Electroporation, Control

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Multiplexed gene knock-in in human T cells. (A–C) Diagrams of nanoplasmid knock-in constructs are provided on the top. Representative contour plots (left) and bar graphs (right) showing the frequency of CD8 + T cells expressing mNG (A) 10 d after electroporation with a nanoplasmid TRAC -mNG donor template and Cas9-RNPs targeting the TRAC locus, mCherry (B) 10 d after electroporation with a nanoplasmid TRAC -mCherry donor template and Cas9-RNPs targeting the TRAC locus, or either mNG or mCherry (C) 10 d after electroporation with two nanoplasmid donor templates ( TRAC -mNG and TRAC -mCherry) and Cas9-RNPs targeting the TRAC locus. Graph on the right for C indicates proportion of transgene expressing cells that express mNG (green), mCherry (red), or both (blue). Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed three times. (D–F) Diagrams of nanoplasmids used in dual targeting study, RAB11A -YFP and TRAC -mCherry (D); representative contour plot (E) showing the frequency of CD8 + T cells expressing YFP, mCherry, or both; and bar graphs (F) showing knock-in efficiency, cell viability, and total cell recovery of CD8 + T cells 10 d after electroporation with nanoplasmid donors RAB11A -YFP and TRAC -mCherry and Cas9-RNPs targeting the RAB11A and TRAC loci. (G) Proportion of transgene expressing T cells co-transfected with nanoplasmid donors RAB11A -YFP and TRAC -mCherry and Cas9-RNPs targeting the RAB11A and TRAC loci that express YFP (green), mCherry (red), or both (blue). Circles represent individual donors, and bars represent median values with range ( n = 4). This experiment was performed three times. (H) Diagrams of nanoplasmids used in dual targeting study, AAVS1 -mNG and TRAC -mCherry. (I and J) Representative contour plot showing the frequency of CD8 + T cells expressing mNG, mCherry or both (I) and bar graphs (J) showing knock-in efficiency, cell viability, and total cell recovery of CD8 + T cells 10 d after electroporation with nanoplasmid donors AAVS1 -mNG and TRAC -mCherry and Cas9-RNPs targeting the AAVS1 and TRAC loci. (K) Proportion of transgene expressing cells co-transfected with nanoplasmid donors AAVS1 -mNG and TRAC -mCherry and Cas9-RNPs targeting the AAVS1 and TRAC loci that express mNG (green), mCherry (red), or both (blue). Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. *, P < 0.05; **, P < 0.01 in RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Gene Knock-In, Knock-In, Construct, Expressing, Electroporation, Cell Recovery, Transfection

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Multiplexed gene knock-in in human T cells. (A–C) Diagrams of pUC57 plasmid knock-in constructs are provided on the top. Representative contour plots (left) and bar graphs (right) showing the frequency of CD8 + T cells expressing mNG (A) 10 d after electroporation with a pUC57 plasmid TRAC -mNG donor template and Cas9-RNPs targeting the TRAC locus ( n = 3), mCherry (B) 10 d after electroporation with a pUC57 plasmid TRAC -mCherry donor template and Cas9-RNPs targeting the TRAC locus ( n = 3), or either mNG or mCherry (C) 10 d after electroporation with two pUC57 plasmid donor templates ( TRAC -mNG and TRAC -mCherry) and Cas9-RNPs targeting the TRAC locus ( n = 3). Graph on the right for C indicates proportion of transgene expressing cells that express mNG (green), mCherry (red), or both (blue). Circles represent individual donors; bars represent median values with range. This experiment was performed three times. (D) Diagrams of pUC57 plasmids used in dual targeting study, RAB11A -YFP and TRAC -mCherry. (E and F) Representative contour plot showing the frequency of CD8 + T cells expressing YFP, mCherry or both (E) and bar graphs (F) showing knock-in efficiency, cell viability, and total cell recovery of CD8 + T cells 10 d after electroporation with pUC57 donors RAB11A -YFP and TRAC -mCherry and Cas9-RNPs targeting the RAB11A and TRAC loci. (G) Proportion of transgene expressing cells co-transfected with pUC57 donor templates RAB11A -YFP and TRAC -mCherry and Cas9-RNPs targeting the RAB11A and TRAC loci that express YFP (green), mCherry (red), or both (blue). Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed three times. *, P < 0.05; **, P < 0.01 in RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Gene Knock-In, Plasmid Preparation, Knock-In, Construct, Expressing, Electroporation, Cell Recovery, Transfection

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Nonviral CRISPR gene editing with large payloads. (A) Diagram of nanoplasmid knock-in constructs TRAC _NotchICD_mNG, TRAC_ NotchICD_1G4, and TRAC _THEMIS_1G4. (B, D, and F) Representative contour plots showing the frequency of CD8 + T cells expressing mNG (B) or 1G4 TCR (D and F) 5 d after electroporation of a NotchICD_mNG (B), NotchICD_1G4 (D), or THEMIS_1G4 (F) nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus. (C, E, and G) Bar graphs showing the frequency of CD8 + T cells expressing mNG (C) or 1G4 TCR (E and G) and cell viability 5 d after electroporation of a NotchICD_mNG (C), NotchICD_1G4 (E), or THEMIS_1G4 (G) nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors, and bars represent median values with range ( n = 3). This experiment was performed three times. *, P < 0.05; **, P < 0.01 in paired t test.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: CRISPR, Knock-In, Construct, Expressing, Electroporation

Journal: Stem Cell Research & Therapy

Article Title: Therapeutic impact of human trophoblast stem cells in peritoneal and pneumonia-induced sepsis in mice

doi: 10.1186/s13287-025-04479-z

Figure Lengend Snippet: Characterization of CD117 + hTSCs in vitro. (a) Representative image of human placental chorionic villi. (b) Chorionic villi stained positive for CD117 (green), DAPI for nuclear staining (blue). Scale bar presents 10 μm. (c & d) hTSCs isolated from the villi express CDX2 (red, c ), a marker for trophoblast stem cells, and CD117 (green, d ), DAPI for nuclear staining (blue). Scale bars present 50 μm. (e) A hTSC clone stained positive for CD117 in limited dilution culture. Multiple images stitched. (f) Representative scatter plots of flow cytometer for CD73 versus CD90 (upper left panel), CD105 versus CD90 (upper middle panel), CD31 versus CD11b (lower left panel) and CD34 versus CD45 (lower right panel). HLA I versus HLA II (upper right panel). Quantitation of the flow cytometric assay in the bar graph show percentage of markers for MSCs (CD90, CD73, CD105) and hematopoietic cells (CD34, CD31, CD45 and CD11b), and HLA I (HLA-A, HLA-B and HLA-C) and HLA II (HLA II: HLA-DR, HLA-DP and HLA-DQ) in the total hTSC population, n = 3 for each marker

Article Snippet: The subpopulation of CD117 + cytotrophoblast cells (hTSCs) were isolated using anti-human CD117 MicroBeads (Miltenyi Biotec, #130-091-332) by MACS separation system and were expanded in DMEM/F12 (Lonza, #12–719 F) medium supplied with 10% FBS, 25 nG/mL hFGF (PeproTech, #100-18B) and 1% Penicillin/Streptomycin [ , ].

Techniques: In Vitro, Staining, Isolation, Marker, Flow Cytometry, Quantitation Assay

Journal: Nature

Article Title: TGFβ links EBV to multisystem inflammatory syndrome in children

doi: 10.1038/s41586-025-08697-6

Figure Lengend Snippet: a , b , GSEA using a previously defined TGFβ gene set applied to T cells ( a ) and the gene set ‘Li M200 antigen processing and presentation’ applied to monocyte clusters ( b ) depicted as both UMAP (left) and a dot plot (right). c , Schematic overview of T cell reactivation assays. d , Frequencies of overall activated (CD69 + ) and antigen-specific reactivated (CD137 + CD69 + and CD154 + CD69 + ) CD4 + or CD8 + memory T cells (T mem , CD45RO + ) from patients with MIS-C during the acute phase and at follow-up after symptoms resolved ( n = 8 patients and n = 5 different viral peptides). e , f , Frequencies of overall activated and antigen-specific reactivated cells of CD4 + and CD8 + memory T cells (T mem ; CD45RO + ) from healthy donors ( n = 6) treated with serum from patients with MIS-C ( e ; n = 7) or patients with severe COVID-19 ( f ; n = 5) with or without anti-TGFβ. Samples that were obtained more than 24 h after the start of treatment are colour-coded in yellow. Unpaired ( a , b ) or paired ( d – f ) two-tailed Mann–Whitney U -tests.

Article Snippet: After stimulation, cells were stained with TotalSeq anti-human Hashtags as previously mentioned, followed by CD154 MACS enrichment according to the manufacturer’s protocol (CD154 MicroBead Kit, human; Miltenyi Biotec).

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: TGFβ links EBV to multisystem inflammatory syndrome in children

doi: 10.1038/s41586-025-08697-6

Figure Lengend Snippet: a , Gating strategy used in flow cytometry of the T cell reactivity assays depicted in Fig. . Cells were identified by size and granularity in a FSC-vs SSC plot, followed by doublet exclusion in an FSC-A vs. FSC-H plot. Dump + (DAPI, CD14 and CD19) + cells were also excluded. As CD3 is downregulated after T cell activation (SEB plot in second row), the gate was extended to include CD3 low CD45RO + cells. CD4 + epitope-specific T cells were identified as CD69 + CD154 + and CD8 + epitope specific T cells were identified as CD69 + CD137 + or as CD69 + CD154 + . SEB was used as a positive control for correct gating. b , Cell counts for CD69 + or CD69 + and CD154 + or CD137 + memory T cells from Fig. . c , Gating strategy used in flow cytometry of the T cell reactivity assays depicted in d. d , Frequencies of overall activated and antigen-specific reactivated cells of CD4 + and CD8 + memory T cells from six children with a confirmed infection with SARS-CoV-2 during the acute phase and follow-up upon after resolution of symptoms. e , Frequencies of overall activated and antigen-specific reactivated cells of CD4 + and CD8 + memory T cells from healthy donors ( n = 6) treated with 50 ng ml −1 TGFβ1. f , Frequencies of TCRVβ21.3 + on total T cells were quantified by Flow cytometry over time after treatment start with IVIG and methylprednisolone. Horizontal lines indicate normal range (0.9-4.9% for CD8 + T cells; 1.5-4-7% for CD4 + T cells) of TCRVβ21.3 + T cells ( n = 25, children with MIS-C). g , Significantly regulated TRBV determined by TCR sequencing of activated T cells. Dots indicate the frequency of specific TRBV in each sample relative to all TCRs sequenced. h , Frequencies of TRAV gene associated to TRBV11-2 + T cells not depicted in Fig. . i , HLA-class I haplotyping and ( j-k ) HLA-class-II haplotyping of our MIS-C cohort ( n = 20 patients and n = 10 healthy controls including the 4 children used as a control for the scRNAseq experiments). Additionally, HLA-haplotyping from a previously published MIS-C cohort ( n = 7 patients and 9 controls) was included. l-m , Sorting strategy for Fig. . P -values for ( b + d-e + h ) were determined by paired two-tailed Mann-Whitney- U -tests.

Article Snippet: After stimulation, cells were stained with TotalSeq anti-human Hashtags as previously mentioned, followed by CD154 MACS enrichment according to the manufacturer’s protocol (CD154 MicroBead Kit, human; Miltenyi Biotec).

Techniques: Flow Cytometry, Activation Assay, Positive Control, Infection, Sequencing, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: TGFβ links EBV to multisystem inflammatory syndrome in children

doi: 10.1038/s41586-025-08697-6

Figure Lengend Snippet: a , Schematic showing generation of virus-specific TCR libraries and comparison of virus-specific TCRs with MIS-C-specific TCRs. scTCR-seq, single-cell TCR sequencing. b , UMAP of 22,344 virus-specific T cells from donors restimulated with EBV ( n = 5), CMV ( n = 5), SARS-CoV-2 ( n = 3) or measles ( n = 3) peptides, representing 18,010 sequenced TCRβ chains and 15,496 full TCRs. AdV-specific T cells were TCR-sequenced. Virus-specificities are colour-coded. c , TRVB11-2 + T cells superimposed on the UMAP in b . d , Gene expression superimposed on the UMAP of antigen-specific T cells, showing that most TRVB11-2 + T cells have a CD4 or CD8 cytotoxic phenotype (low: ICOS ; high: PRF1 , GZMB , LAMP1 ). e , TCR repertoires of EBV ( n = 5), CMV ( n = 5), SARS-CoV-2 ( n = 3), measles ( n = 3) and AdV ( n = 1) virus-specific T cells from healthy donors, analysed by ARTE . Heat map showing distribution of TRAV gene expression associated with TRBV11-2 -positive T cells in virus-specific and MIS-C T cells ( n = 11) T cells, compared with 6 wpi no MIS-C ( n = 4) and paediatric influenza ( n = 3) T cells. Unsupervised clustering was performed with the R package pheatmap. f , TCRVβ21.3 expression on memory T (T mem ) cells after stimulation with EBNA2 275–294 (left) or EBNA2 279–289 (right) peptides, analysed by ARTE. Frequencies of TCRVβ21.3 + in all CD4 + (top) and CD8 + (bottom) memory T cells and those with antigen-specific reactivation (CD154 + CD69 + ) from n = 7 donors. Flow cytometry gating is shown in Extended Data Fig. . Two-sided paired t -test.

Article Snippet: After stimulation, cells were stained with TotalSeq anti-human Hashtags as previously mentioned, followed by CD154 MACS enrichment according to the manufacturer’s protocol (CD154 MicroBead Kit, human; Miltenyi Biotec).

Techniques: Virus, Comparison, Sequencing, Gene Expression, Expressing, Flow Cytometry

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (a) HepG2 with or without various treatments for 24 hours and stable HepG2 cells that expressed a control shRNA (sh-Ctrl) or the Atg5 shRNA (sh-Atg5) were subjected to flow cytometry analysis for CD133+ cells. Results represent the mean ± SEM of three independent experiments. None, no treatment. (B) HepG2 cells with the treatments shown in (A) were lysed for immunoblot analysis. LC3-I, non-lipidated LC3; LC3-II, lipidated LC3. The β-actin protein was also analyzed to serve as the loading control. (C) Sphere-formation assay of CD133+ and CD133− HepG2 cells. The panels shown to the left are representative results of spheres formed by CD133+ and CD133− HepG2 cells with and without stable ATG5 knockdown. Scale bar=200 μm. The histogram shown to the right indicated the number of spheres larger than 100 μm in diameter when 500 CD133+ cells were seeded. The results represent the mean ± SEM of three independent experiments. (D) HepG2 cells with various treatments for 24 hours were incubated with MicroBeads (Miltenyi Biotec) for the isolation of CD133+ cells, which were then analyzed for their sphere-forming ability. 500 cells were seeded for the assay. Also see Figure S1.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Control, shRNA, Flow Cytometry, Western Blot, Tube Formation Assay, Knockdown, Incubation, Isolation

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) Hep3B and Huh7 cells with various treatments for 24 hours were subjected to flow cytometry analysis for CD133+ cells. (B) HepG2 and Huh7 cells were transfected with the p53-expressing plasmid for two days, treated with 3-MA or rapamycin for another 24 hours and then subjected to flow cytometry analysis for CD133+ cells. (C) HepG2 cells transfected with the control siRNA (si-Ctrl) or the p53 siRNA (si-p53) for two days or treated with PFTα or DMSO for one day were analyzed for their CD133+ cells by flow cytometry (top panel) or sphere-forming ability of their CD133+ cells (bottom panel). The results shown in (A), (B) and (C) represent the mean ± SEM of three independent experiments. (D) HepG2 cells treated with DMSO or PFTα for one day or with siRNA for two days were lysed for immunoblot analysis. (E) Stable HepG2 cells that expressed control shRNA (sh-Ctrl), sh-Atg5, or both sh-Atg5 and sh-p53 were lysed for immunoblot analysis. (F) Cells mentioned in (E) were used for the sphere-formation assay. Also see Figure S2.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Control, Western Blot, shRNA, Tube Formation Assay

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) HepG2 cells with various treatments for 24 hours or stably expressing the control shRNA or the Atg5 shRNA were lysed for immunoblot analysis. (B) Immunoblot analysis of HepG2 cells transfected with either the control vector or the expression vector for various p53 proteins. Cells were lysed two days after DNA transfection for immunoblot analysis. None, control cells with no DNA transfection. (C) The experiments were conducted the same way as in (B), with the exception that Hep3B cells were used for the expression studies. (D) Hep3B or HepG2 cells were transfected with various p53-expressing plasmids or the control vector as indicated for two days followed by flow cytometry analysis for CD133+ cells. (E) HepG2 cells were transfected with the p53-expressing plasmids for two days, and CD133+ cells were then isolated for the sphere-formation assay. (F) The experiments were conducted the same way as in (E), with the exception that Hep3B cells were used for the studies. The results in (D–F) represented the mean ± SEM of three independent experiments. Also see Figure S3.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Stable Transfection, Expressing, Control, shRNA, Western Blot, Transfection, Plasmid Preparation, Flow Cytometry, Isolation, Tube Formation Assay

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) Top panel, HepG2 cells were treated with DMSO, Mdivi-1 or CCCP for one day and then subjected to flow cytometry analysis for CD133+ cells; bottom panel, CD133+ HepG2 cells were isolated and treated with Mdivi-1 or CCCP for two days and then analyzed for their sphere-forming ability. (B) HepG2 cells without treatment or with the treatment of DMSO, CCCP or Mdivi-1 for one day were lysed for immunoblot analysis. Cells were also subjected to subcellular fractionation for the isolation of mitochondria, cytosol, and nuclei for immunoblot analysis. Tom20, β-actin and lamin B1 were used as the loading controls for mitochondria, cytosol and nucleus, respectively, to ensure equal amount of proteins were loaded on the gel. (C) Confocal microscopy for the analysis of the subcellular localization of p53(pS392) in HepG2 cells treated with DMSO, Mdivi-1 or CCCP. TOM20 was used as the marker for mitochondria. The areas boxed are enlarged at the bottom. Scale bar, 10 μm. (D) The results shown in (C) were quantified with a Leica TCS SP8 fluorescent confocal microscope. The results indicated the percentages of p53(pS392) that colocalized with TOM20. The results represent the mean ± SEM of at least 30 cells that were analyzed. See also Figure S5.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Flow Cytometry, Isolation, Western Blot, Fractionation, Confocal Microscopy, Marker, Microscopy

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) Effects of PINK1 knockdown on CD133+ HepG2 cells (top panel), their sphere-forming ability (middle panel) and their effects on the Nanog promoter using the Nanog-luc1 reporter (bottom panel). HepG2 cells transfected with either the control siRNA or the PINK1 siRNA for two days were analyzed. In the bottom panel, HepG2 cells were also transfected with the Nanog-luc1 reporter (see Figure 3B) for the analysis of luciferase activity. The luciferase activity of cells without the transfection of siRNA was arbitrarily defined as 1. The results represented the mean ± SEM of three independent experiments. (B) Effects of PINK1 over-expression on CD133+ HepG2 cells (top panel), their sphere-forming ability (middle panel) and their effects on the Nanog promoter (bottom panel). The experiments were conducted the same way as in (A), except that instead of using siRNA, cells were transfected with either the control vector or the PINK1-expressing plasmid. (C) Immunoblot analysis of HepG2 cells with PINK1 knockdown (left panels) or PINK1 over-expression (right panels) were lysed for immunoblot analysis. Total cell lysates as well as the nuclear lysates (bottom two panels) were analyzed. (D) PINK1 in HepG2, Hep3B or Huh7 cells was immunoprecipitated with a control antibody (−) or the anti-PINK1 antibody (+) and then incubated with GST-p53 in the presence of ATP. The GST-p53 phosphorylated at S392 was analyzed using the anti-p53 antibody that recognized phosphoserine-392. GST-p53 added in the reaction and PINK1 immunoprecipitated were also analyzed by immunoblot (bottom two panels). Numbers to the left of the top panel indicate protein molecular weight markers. (E) GST-p53 was mixed with GST-PINK1 or GST and incubated in the presence of ATP. The phosphorylation of p53 at S392 was then analyzed with the antibody that recognized phosphoserine-392. GST-p53, GST-PINK1 and GST used for the reaction was also analyzed by anti-p53, anti-PINK1 and anti-GST antibodies, respectively (bottom three panels). Numbers to the left indicate protein molecular weight markers. (F) Co-immunoprecipitation of p53 and p53(pS392) with PINK1. HepG2 cells were lysed and immunoprecipitated using the anti-PINK1 antibody or the control antibody followed by immunoblot analysis for p53, p53(pS392) and PINK1. (G) Co-immunoprecipitation of p53 and p53(pS392) with PINK1 using the anti-PINK1 antibody in different subcellular fractions (top 3 panels). β-actin, lamin B1 and Tom20 were used as the markers for cytosolic (C), nuclear (N) and mitochondrial (M) fractions. Equal amounts of p53 were used for the co-immunoprecipitation experiment (bottom 2 panels). See also Figure S6.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Knockdown, Transfection, Control, Luciferase, Activity Assay, Over Expression, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Incubation, Molecular Weight, Phospho-proteomics

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Virus, Recombinant, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Mouse Assay, Mutagenesis, Software, Imaging, Extraction, Isolation, DNA Labeling

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Increased functional potency of multi-edited CAR-T cells manufactured by a non-viral transfection system

doi: 10.1016/j.omtm.2024.101389

Figure Lengend Snippet: Analysis of TSCM phenotype over time (A and B) CD4 + (A) and (B) CD8 + T cells were expanded for 0–4 days post-transfection in a 24-well G-Rex plate and stimulation with TransACT and IL-2 ( N = 3 donors in duplicate). T SCM phenotype (CD45RA + CD45RO − CD62L + CCR7 + CD95 + ) was measured by flow cytometry, and a comparison of results for Solupore and electroporation TKO transfected T cells and untransfected control cells is shown. (C and D) CAR-T cells were generated via lentiviral transduction, and the T SCM phenotype of CD4 + and CD8 + CAR-T cells was analyzed 24 h post-transduction (pre-cryopreservation; C) and (D) post-thaw. n = 3 donors, paired t test, p = 0.0396 (C). Data represented as mean +/- SD.

Article Snippet: T cells were isolated from the fresh Leukopak, within 24 h of collection, via positive selection using the Straight from Leukopak CD4 (Miltenyi Biotec, 130117022) and CD8 (Miltenyi Biotec, 130117019) Microbead kits and MultiMACS Cell24 Separator Plus (Miltenyi Biotec 130-098-637), according to the manufacturer’s protocol.

Techniques: Transfection, Flow Cytometry, Comparison, Electroporation, Control, Generated, Transduction

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Increased functional potency of multi-edited CAR-T cells manufactured by a non-viral transfection system

doi: 10.1016/j.omtm.2024.101389

Figure Lengend Snippet: Flow cytometry antibodies

Article Snippet: T cells were isolated from the fresh Leukopak, within 24 h of collection, via positive selection using the Straight from Leukopak CD4 (Miltenyi Biotec, 130117022) and CD8 (Miltenyi Biotec, 130117019) Microbead kits and MultiMACS Cell24 Separator Plus (Miltenyi Biotec 130-098-637), according to the manufacturer’s protocol.

Techniques: Flow Cytometry, In Vivo

Journal: PLoS ONE

Article Title: Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells

doi: 10.1371/journal.pone.0129658

Figure Lengend Snippet: Purified CD19 + B cells were labeled with CFSE, stimulated with anti-IgM, anti-CD40 mAb and IL-21 (BCR method) in the absence (control; CTRL) or presence of TAC (6ng/ml) or SRL (2ng/ml or 6ng/ml) and flow cytometric analyses were performed after 6 days in culture. (A) A representative experiment: cells were gated on viable lymphocytes and analyzed for CFSE diluting proliferating cells. This scheme of analysis was used in all subsequent experiment, unless indicated otherwise. (B) The percentage of proliferating CD19 + B cells as obtained in A from 7 different experiments. (C) Absolute number of proliferating CD19 + B cells was calculated in each experiment by multiplying the recovered cell counts with the percentage of proliferating cells as in A (n = 7). Statistically significant (*p < 0.05) inhibition of B cell proliferation was observed with SRL at both subtherapeutic (2ng/ml) and therapeutic (6ng/ml) doses.

Article Snippet: Total B cells were isolated from PBMC by positive selection using human CD19 MicroBeads kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Labeling, Control, Inhibition

Journal: PLoS ONE

Article Title: Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells

doi: 10.1371/journal.pone.0129658

Figure Lengend Snippet: Purified CD19 + B cells were stimulated with anti-IgM, anti-CD40 mAb and IL-21 and multi-color flow cytometric analyses were performed on day 6 as described in . The figures A , B and C show the expression of various surface markers on stimulated B cells (Percentage of positive cells/total proliferating CD19 + cells) p < 0.05, **p < 0.01. CTRL, control; TAC6, 6 ng/ml TAC; SRL2, 2 ng/ml SRL; SRL6, 6 ng/ml SRL.

Article Snippet: Total B cells were isolated from PBMC by positive selection using human CD19 MicroBeads kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Expressing, Control

Journal: PLoS ONE

Article Title: Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells

doi: 10.1371/journal.pone.0129658

Figure Lengend Snippet: B cells were purified by depleting non-B cells resulting in >95% CD19 + cells which subsequently sorted into CD27 − (naïve) and CD27 + (memory) B-cell fractions. These subsets were labeled with CFSE, stimulated with anti-IgM, anti-CD40 mAb and IL-21 in the absence (CTRL) or presence of TAC or SRL at 6ng/ml and were analyzed by multicolor flow cytometry after 6 days in culture. (A) A representative experiment: histogram plots show dilution of CFSE in the proliferating CD19 + CD27 − (upper panel) or CD19 + CD27 + (lower panel) cells. (B) Data are from four different independent experiments are shown as mean ± SD percent proliferating naïve CD19 + CD27 − and memory CD19 + CD27 + B cells. (C) B cell subsets showing indicated surface markers were analyzed and plotted as mean ± SD (n = 4) percent of proliferating cells in the cultures of naïve CD19 + CD27 − (upper panel) and memory CD19 + CD27 + B cells (lower panel). The residual cells that proliferated in presence of SRL demonstrated an activated phenotype. *p < 0.05, **p < 0.01.

Article Snippet: Total B cells were isolated from PBMC by positive selection using human CD19 MicroBeads kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Labeling, Flow Cytometry

Journal: PLoS ONE

Article Title: Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells

doi: 10.1371/journal.pone.0129658

Figure Lengend Snippet: Purified CD19 + B cells were cultured as in Figs and . (A) A representative experiment showing flow cytometric profile indicative of putative plasma cells (CD19 low ) in proliferated B cells (gated on viable lymphocytes; see ). (B) The mean ± SD percentage of such CD19 low B cells from 4 different independent experiments. (C) Mean ± SD (n = 4) percentage of CD19 low CD38 ++ plasmablasts, CD19 low CD138 + plasma cells, Blimp1 + PAX5 - cells and CD138 + Blimp1 + cells in the proliferating CD19 low cells. *p < 0.05, **p < 0.01. CTRL, control; TAC, 6 ng/ml TAC; SRL, 6 ng/ml SRL.

Article Snippet: Total B cells were isolated from PBMC by positive selection using human CD19 MicroBeads kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Cell Culture, Clinical Proteomics, Control

Journal: PLoS ONE

Article Title: Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells

doi: 10.1371/journal.pone.0129658

Figure Lengend Snippet: Purified CD19 + B cells were pre-stimulated for 6 days with anti-IgM, anti-CD40 mAb and IL-21 in the absence (CTRL) or presence of 6ng/ml TAC or SRL. These cultured B cells were used as stimulators in 6-day MLRs of allogeneic CFSE-labelled CD4 + CD25 − T cell responders. ( A ) Level of proliferation differentially induced by pre-cultured B cells as detected by CFSE dilution in the allogeneic CD4 responder cells (representative experiment on the left, and compiled data from 8 independent experiments on the right). ( B ) Percentage of responding T cells positive for memory marker (CD45RO) and activation markers (CD62L, CD25, CD69, CD95) after co-culture with pre-stimulated B cells (n = 8); ( C ) Mean ± SD (n = 4) percentage of responding proliferating T cells expressing intracellular cytokines (top row) and transcription factors (bottom row). Taken together the data indicated that B cells that proliferated in presence of SRL on a per cell basis were capable of inducing alloreactive T cell proliferation towards a Th1 phenotype. *p < 0.05. ** p < 0.01.

Article Snippet: Total B cells were isolated from PBMC by positive selection using human CD19 MicroBeads kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Cell Culture, Marker, Activation Assay, Co-Culture Assay, Expressing