Journal: PLoS ONE

Article Title: High Specificity in Circulating Tumor Cell Identification Is Required for Accurate Evaluation of Programmed Death-Ligand 1

doi: 10.1371/journal.pone.0159397

Figure Lengend Snippet: PBMCs isolated from patients with NSCLC and depleted of CD45 + cells were stained for flow cytometric evaluation. (A) Cells were gated first on live cell populations, then on cells which were either CD11b + CD45 lo or CD11b - CD45 - . (B) Subsequent plots displayed previously gated CD11b + CD45 lo cells (red) over all other live cells (black) on X and Y axes of FSC vs. SSC, (C) CD45 vs. pCK, or (E) CD45 vs. the isotype control for pCK, IgG1. (D) This staining and gating strategy was performed over three different patient samples to evaluate staining intensity, or mean fluorescence intensity (MFI), of pCK or (F) the isotype control. Significance was evaluated by one-tailed, paired T-tests with significance considered at p<0.05 (*).

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were incubated with commercially available, pre-conjugated, anti-CD45 magnetic beads (Miltenyi) in a buffer containing 2 mM EDTA (Fisher Scientific) and 0.5% bovine serum albumin (BSA, Sigma-Aldrich) in phosphate buffered saline (PBS, Hyclone), after which CD45 + cells were removed using magnetic LS MACS columns (Miltenyi).

Techniques: Isolation, Staining, Fluorescence, One-tailed Test

Journal: PLoS ONE

Article Title: High Specificity in Circulating Tumor Cell Identification Is Required for Accurate Evaluation of Programmed Death-Ligand 1

doi: 10.1371/journal.pone.0159397

Figure Lengend Snippet: Cells were isolated from NSCLC patient samples using an antibody against MUC1. (A) Cells were then stained and imaged with a fluorescence microscope and crops of representative 10x images were given as examples of CD11b + CD45 lo or CD11b - CD45 + cells. (B) Cells were identified and analyzed in JEX, then graphed in R with each cell represented as one point. Cells were displayed on an X-axis of mean fluorescence intensity (MFI) of CD45 staining, and a Y-axis of cell size. CD45 + cells (red points) were excluded by manual gating on clustered populations. (C) Subsequent gating in R displayed previously excluded cells (blue) with newly excluded cells (red) and positively selected cells (green) on X- and Y-axes of CD45 and CD11b MFI, respectively. Gating in this plot was based on exclusion of the population of cells which emerged from additional CD11b staining. (D) Cells were isolated from one NSCLC patient sample in three parallel experiments using either an antibody against EpCAM, MUC1, or Vim. The average frequency of CD11b + cells isolated from different antibodies was 22, 27 and 47%, respectively. (E) Results from five different patients were compared using one-tailed, paired T-tests with significance considered at p<0.05 (*).

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were incubated with commercially available, pre-conjugated, anti-CD45 magnetic beads (Miltenyi) in a buffer containing 2 mM EDTA (Fisher Scientific) and 0.5% bovine serum albumin (BSA, Sigma-Aldrich) in phosphate buffered saline (PBS, Hyclone), after which CD45 + cells were removed using magnetic LS MACS columns (Miltenyi).

Techniques: Isolation, Staining, Fluorescence, Microscopy, One-tailed Test

Journal: PLoS ONE

Article Title: High Specificity in Circulating Tumor Cell Identification Is Required for Accurate Evaluation of Programmed Death-Ligand 1

doi: 10.1371/journal.pone.0159397

Figure Lengend Snippet: CTCs were captured from CD45 depleted buffy coats with MUC1 or EpCAM labelled PMPs, then stained and identified in JEX and R based on CD45 and pCK staining. (A) pCK - CD45 + cells were excluded in the first gate (red dots), while pCK + CD45 - cells were positively selected (green dots) as traditionally identified CTCs. (B) Subgated plots enabled further exclusion of CD11b + cells (red dots), from CD11b - cells positively selected as CTCs (green dots) displayed over previously excluded cells (blue dots). Red dots in this subgate represent false-positives. (C) Example 40x images were shown for a WBC (CD11b - CD45 + pCK - ), a CTC false-positive (CD11b + CD45 lo pCK + ), and a true CTC (CD11b - CD45 - pCK + ). (D) Data from multiple samples were graphed as individual data points to demonstrate the frequency of false-positives using either EpCAM or MUC1 capture antibodies. Results from four different patients were compared using a two-tailed, paired T-test.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were incubated with commercially available, pre-conjugated, anti-CD45 magnetic beads (Miltenyi) in a buffer containing 2 mM EDTA (Fisher Scientific) and 0.5% bovine serum albumin (BSA, Sigma-Aldrich) in phosphate buffered saline (PBS, Hyclone), after which CD45 + cells were removed using magnetic LS MACS columns (Miltenyi).

Techniques: Staining, Two Tailed Test

Journal: PLoS ONE

Article Title: High Specificity in Circulating Tumor Cell Identification Is Required for Accurate Evaluation of Programmed Death-Ligand 1

doi: 10.1371/journal.pone.0159397

Figure Lengend Snippet: (A) CTCs captured with MUC1 and identified from patient 324 as either pCK + CD45 - or CD11b - pCK + CD45 - were graphed as individual data points and compared for average PD-L1 MFI and (C) frequency of PD-L1 + CTCs. (B) Data from seven different patient samples were represented as seven single data points and compared by CTC identification criteria, either with or without CD11b exclusion criteria, for average PD-L1 expression and (D) frequency of PD-L1 expression on CTCs. Results were compared using two-tailed T-tests; unpaired for each individual sample, and paired for overall comparisons.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were incubated with commercially available, pre-conjugated, anti-CD45 magnetic beads (Miltenyi) in a buffer containing 2 mM EDTA (Fisher Scientific) and 0.5% bovine serum albumin (BSA, Sigma-Aldrich) in phosphate buffered saline (PBS, Hyclone), after which CD45 + cells were removed using magnetic LS MACS columns (Miltenyi).

Techniques: Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Inosine Induces Stemness Features in CAR T cells and Enhances Potency

doi: 10.1101/2023.04.21.537859

Figure Lengend Snippet: A. Flow cytometric analysis of exhaustion marker expression in gated CD19.28z-CAR + (CD19) (blue) and HA.28z-CAR + (HA) T cells (red) at D0 (grey) and at each indicated time-point post-activation. Representative histogram shown of n = 3 donors. B. (Left) Representative flow cytometry contour plot of CD39 expression in HA-CAR − vs. CAR + T cells on day 10 post-activation. (Right) Percent of CD39 + cells in HA-CAR− vs. CAR + T cells from n=12 donors. P values determined by paired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 C. (Left) Representative flow cytometry analysis of CD39 expression in CD8 + vs. CD4 + HA-CAR T cells 10 days post-activation. (Right) Percent of CD39 + cells in CD4 + vs. CD8 + HA-CAR T cells from n = 11 donors. P values determined by paired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 D. Luminex analysis of cytokines secreted by CD39 + vs. CD39 − CD8 + HA-CAR T cells sorted on day 14 and stimulated with Nalm6-GD2 for 24hrs at 1:1 E:T ratio. Data are mean ± s.e.m. of n= 3 donors. P values determined by ratio paired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 E. Gene Set Enrichment Analysis (GSEA) of bulk RNA-seq collected 14 days post-activation of CD39 + compared to CD39 − HA-CAR T cells using the publicly available GSE25087 gene collection. F. CyTOF analysis of CD4 + (bottom) and CD8 + (top) HA-CAR T cells gated based on CD39 expression 10 days post-activation. Heat map represents expression of the indicated markers as median Arcsinh relative to the total CD4 + or CD8 + sample. Representative donor shown of n=3 donors. G. Schematic of immune suppression mediated by purinergic pathway. ATP- adenosine triphosphate; ADP- adenosine diphosphate; AMP- adenosine monophosphate; ADO- adenosine (red); A2aR- adenosine 2a receptor; CD39− ecto-ATP diphosphohydrolase-1; CD73– 5ʹ-ectonucleotidase; NF-κB- Nuclear Factor kappa B. H. (Left) Representative flow cytometry contour plots showing expression of CD39 and CD73 in gated CD4 + or CD8 + HA-, CD19-CAR or mock T cells 14 days post-activation. (Right) Percent of CD39 + CD73 + T cells in CD4 + vs. CD8 + by day 14 HA-, CD19-CAR or mock T cells in 6 donors. P values determined by paired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 I. Concentration of adenosine (ADO) produced by mock or CAR-expressing T cells with or without CD39 or CD73 spiked with 20uM of ATP at day 17 post-activation. Representative data from n=3–5 donors. Average purity of the knock-out cells was > 90%. P values determined by unpaired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 J. (Left) schematic of experimental design of immunosuppression co-culture assays. (Right) Control or A2aR KO CD19.bbz-CAR T cells were activated with Nalm6 at 1:1 ratio in the presence of HA- or CD39KO HA-CAR T cells. IL-2 was measured 24hrs post-stimulation. Data are mean ± s.e.m. from triplicate wells. P values determined by unpaired two-tailed t-tests. Representative result of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Article Snippet: Apheresis of healthy donors was freshly collected and CD4 + and CD8 + T cells were enriched using anti-CD4 and anti-CD8 beads (Miltenyi) and EasySep250 (StemCell).

Techniques: Marker, Expressing, Activation Assay, Flow Cytometry, Two Tailed Test, Luminex, RNA Sequencing Assay, Concentration Assay, Produced, Knock-Out, Co-Culture Assay, Control

Journal: bioRxiv

Article Title: Inosine Induces Stemness Features in CAR T cells and Enhances Potency

doi: 10.1101/2023.04.21.537859

Figure Lengend Snippet: A. Schematic of large-scale manufacturing of GD2.bbz-CAR + T cells in a semi-closed G-Rex system in glucose- or inosine-containing media. 7 days post-activation, transduction efficiency, percentage of viable cells, and CD4/CD8 ratio of GD2 + cells were assessed by flow cytometry. Data are mean+/− s.e.m. from n=2 donors. P values determined by paired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 B. (Left) Expansion of total viable T cells manufactured in control (black) vs. inosine (green)-containing media and (Right) corresponding number of CAR+ cells at day 7. Data are mean+/− s.e.m. from n=2 donors. P values determined by paired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 C. Flow cytometry analysis of the relative frequency of terminally differentiated effector (EMRA; CD45RA + CCR7 − ), naïve (CD45RA + CCR7 + ), central memory (CM; CD45RA − CCR7 + ), and effector memory (EM; CD45RA − CCR7 − ) in CD8 + (top) and CD4 + (bottom) control or inosine-grown GD2-CAR T cells. Representative donor shown. N=2 donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 D. IL-2 and IFNγ secretion by CAR T cells stimulated for 24hrs with 143b, mg63.3 or Nalm6-GD2 tumor lines. Error bars represent mean ± SD of triplicate wells from one representative donor (n=2 donors). P values determined by unpaired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 E. NSG mice were injected with 1 × 10 6 Nalm6 leukemia cells. On D3 post-tumor injection, 2 × 10 6 of mock or GD2.bbz-CAR T cells manufactured in the presence of inosine or in regular media were transferred intravenously. Tumor growth was monitored by bioluminescent imaging. Data are mean ± s.e.m. of n=5 mice per group. P values determined at day 43 by using Mann-Whitney test. Representative results of two independent experiments shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 F. Concentration of CD45 + T cells detected in blood of mice at D33 post-CAR T cell injection. P values determined by unpaired two-tailed t-test with Welch’s correction. Results from one experiment shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 G. Proposed model showing metabolic and epigenetic differences between tonic signaling CAR-T cells expanded in glucose- vs. inosine-containing culture media. Reprograming is due to an increase in glutaminolysis and polyamine synthesis and downregulation of CD73, FOXP3, A2aR and c-AMP response transcription factors. This results in an increase of stemness and a reduction of the immunosuppressive phenotype.

Article Snippet: Apheresis of healthy donors was freshly collected and CD4 + and CD8 + T cells were enriched using anti-CD4 and anti-CD8 beads (Miltenyi) and EasySep250 (StemCell).

Techniques: Activation Assay, Transduction, Flow Cytometry, Two Tailed Test, Control, Injection, Imaging, MANN-WHITNEY, Concentration Assay

Journal: Cell metabolism

Article Title: The translational machinery of human CD4 + T cells is poised for activation and controls the switch from quiescence to metabolic remodelling

doi: 10.1016/j.cmet.2018.08.009

Figure Lengend Snippet: (A) Heat map showing the pairwise Pearson correlation coefficient (PCC) of the log2 read count of all metabolic genes across CD4+ and CD8+ T cell subsets (Pearson correlation coefficient 0.7-1 between cell subsets).

Article Snippet: Human CD4 (VIT-4)-VioGreen , Miltenyi , Cat#130-096-900.

Techniques:

Journal: Cell metabolism

Article Title: The translational machinery of human CD4 + T cells is poised for activation and controls the switch from quiescence to metabolic remodelling

doi: 10.1016/j.cmet.2018.08.009

Figure Lengend Snippet: (A) Radar charts of the distribution of ribosomal genes across CD4+ and CD8+ T cell subsets. In the radar chart T cell subsets are represented on axes starting from the same point and ribosomal capability by a spoke. The length of a spoke is proportional to the magnitude of the ribosomal capability. Naïve and Central Memory have the highest levels of ribosomal mRNAs.

Article Snippet: Human CD4 (VIT-4)-VioGreen , Miltenyi , Cat#130-096-900.

Techniques:

Journal: Cell metabolism

Article Title: The translational machinery of human CD4 + T cells is poised for activation and controls the switch from quiescence to metabolic remodelling

doi: 10.1016/j.cmet.2018.08.009

Figure Lengend Snippet: (A) Flow cytometry plots representative of three independent experiments showing the expression of GLUT1 protein in CD4+ naive T cells or in the same cells activated for 24h, 48h and 72h. Right, quantification of flow cytometry data. Data are mean ± sd. p values are determined by two-tailed Student’s t-tests. * * * P < 0.001.

Article Snippet: Human CD4 (VIT-4)-VioGreen , Miltenyi , Cat#130-096-900.

Techniques: Flow Cytometry, Expressing, Two Tailed Test

Journal: Cell metabolism

Article Title: The translational machinery of human CD4 + T cells is poised for activation and controls the switch from quiescence to metabolic remodelling

doi: 10.1016/j.cmet.2018.08.009

Figure Lengend Snippet: (A) Metabolome analysis of CD4+ T cells collected at the indicated time points after TCR stimulation. The log2 value for each metabolite represents the average of three replicates. Metabolites were clustered in seven categories. Clusters are shown.

Article Snippet: Human CD4 (VIT-4)-VioGreen , Miltenyi , Cat#130-096-900.

Techniques:

Journal: Cell metabolism

Article Title: The translational machinery of human CD4 + T cells is poised for activation and controls the switch from quiescence to metabolic remodelling

doi: 10.1016/j.cmet.2018.08.009

Figure Lengend Snippet: Key Resource Table

Article Snippet: Human CD4 (VIT-4)-VioGreen , Miltenyi , Cat#130-096-900.

Techniques: Recombinant, Cell Isolation, Pyruvate Assay, Reporter Assay, Software

Journal: eLife

Article Title: Notch-induced endoplasmic reticulum-associated degradation governs mouse thymocyte β−selection

doi: 10.7554/eLife.69975

Figure Lengend Snippet: ( A ) Diagram showing two ER quality control machineries: ERAD and UPR. The E3 ubiquitin ligase HRD1 and its adaptor protein SEL1L is the most conserved ERAD complex in mammals. While correctly folded proteins exit the ER, misfolded proteins in the ER are recruited to the SEL1L-HRD1 complex through ER chaperones (such as BiP, EDEM, and OS9), and then retrotranslocated into the cytosol, ubiquitinated and degraded by the proteasome. Failure to clear the misfolded or unfolded proteins in the ER activates the UPR signaling through three ER stress sensors IRE1α, ATF6, and PERK. Upon activation, IRE1α oligomerizes and undergoes trans -autophosphorylation to activate its RNase domain, resulting in the removal of 26 nucleotides from unspliced XBP1 ( XBP1u ) mRNA to produce mature, spliced XBP1 ( XBP1s ) mRNA. PERK is a serine-threonine kinase. ER stress induces PERK-dependent eIF2α phosphorylation and subsequent increased cap-independent translation of ATF4 and induction of CHOP. ( B ) Schematic diagram of T-cell development in the thymus. CLP: common lymphoid progenitors; ETP: early T lineage precursor (Lin - CD4 - CD8 - CD44 + CD25 - CD117 + ); DN2: double negative two thymocytes (Lin - CD4 - CD8 - CD44 + CD25 + ); DN3: double negative three thymocytes (Lin - CD4 - CD8 - CD44 - CD25 + ); DN4: double negative four thymocytes (Lin - CD4 - CD8 - CD44 - CD25 - ); ISP: immature single-positive thymocytes (Lin - CD8 + CD24 + TCRβ - ); DP: double positive thymocytes (Lin - CD4 + CD8 + ); SP4: CD4 single positive thymocytes (Lin - CD4 + CD8 - ); SP8: CD8 single positive thymocytes (Lin - CD4 - CD8 + ). ( C ) Representative pseudocolor plots showing the gating strategy to identify different thymocyte subsets in the thymus.

Article Snippet: Other , CD4 (L3T4) MicroBeads, mouse 1 x 2 mL , 130-117-043 , Miltenyi Biotec , .

Techniques: Control, Ubiquitin Proteomics, Activation Assay, Phospho-proteomics

Journal: eLife

Article Title: Notch-induced endoplasmic reticulum-associated degradation governs mouse thymocyte β−selection

doi: 10.7554/eLife.69975

Figure Lengend Snippet: ( A ) Schematic of labeling and detection of nascent protein with OP-Puro. OP-Puro (O-propargyl puromycin) is a cell-permeable puromycin analog that is incorporated into the C-terminus of newly synthesized peptide chain. Fluorophore conjugated with Alexa Fluor 647 was then attached to OP-Puro through a copper-catalyzed click chemistry reaction between alkyne and azide group, which quantifies protein synthesis by fluorescence intensity. ( B ) Representative histogram (left) and quantification (right) of OP-Puro incorporation in different thymocyte subsets from 8-week-old wild-type mice. FMO represents AF647 control which is the background from the click chemistry in the absence of OP-Puro. MFI, mean fluorescence intensity. n = four mice. ( C ) Quantification of tetraphenylethene maleimide (TMI) fluorescence in different thymocyte subsets from 8-week-old wild-type mice. n = seven mice. ( D ) Quantitative RT-PCR analysis of ERAD ( Sel1l ) and UPR-related ( Xbp1 , Hspa5(Bip) , Dnajb9 , Ddit3 ( Chop ), Atf4 ) genes expression in different thymocyte subsets from 6-week-old wild-type mice. Data are presented relative to Actb; n = three mice. ( B–D ), ETP: early T lineage precursor (Lin - CD4 - CD8 - CD44 + CD25 - CD117 + ); DN2: double negative two thymocytes (Lin - CD4 - CD8 - CD44 + CD25 + ); DN3: double negative three thymocytes (Lin - CD4 - CD8 - CD44 - CD25 + ); DN4: double negative four thymocytes (Lin - CD4 - CD8 - CD44 - CD25 - ); DP: double positive thymocytes (Lin - CD4 + CD8 + ); SP4: CD4 single positive thymocytes (Lin - CD4 + CD8 - ); SP8: CD8 single positive thymocytes (Lin - CD4 - CD8 + ). Results are shown as mean ± s.d. The statistical significance was calculated by one-way ANOVA with Bonferroni test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant. Figure 1—source data 1. Excel file containing numerical values shown in .

Article Snippet: Other , CD4 (L3T4) MicroBeads, mouse 1 x 2 mL , 130-117-043 , Miltenyi Biotec , .

Techniques: Labeling, Synthesized, Fluorescence, Control, Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: Notch-induced endoplasmic reticulum-associated degradation governs mouse thymocyte β−selection

doi: 10.7554/eLife.69975

Figure Lengend Snippet: ( A ) Representative flow cytometry plots of different thymocyte subsets in control (Ctrl, Sel1l flox/flox ) and Sel1l CKO ( Sel1l flox/flox ; CD2 -iCre) mice. ( B ) Diagram showing different stages of CD2 -iCre and Cd4-Cre initiated gene depletion during T cell development. ( C ) Quantitative RT-PCR analysis of Sel1l in different thymocyte subsets from control (Ctrl, Sel1l flox/flox ) and Sel1l flox/flox ; Cd4- Cre mice. Data are presented relative to Actb. n = 3. ( D and E ) Representative images of thymus ( D ) and quantification of thymus cellularity ( E ) in 6- to 8-week-old control (Ctrl, Sel1l flox/flox ) and Sel1l flox/flox ; Cd4- Cre mice. n = 3. ( F and G ) Quantification of cell numbers of different thymocyte subsets from control (Ctrl, Sel1l flox/flox ) and Sel1l flox/flox ; Cd4 -Cre mice. n = 3. ( H ) Percentage of Ctrl or Sel1l CKO donor-derived progenitors in the bone marrow of recipient mice 14 weeks after transplantation. n = 4–5. ( I ) Quantification of Ctrl or Sel1l CKO donor-derived DN3/DN4 ratio. n = 4–5. ( J ) Percentage of Ctrl or Sel1l CKO donor-derived CD4 + T cells, CD8 + T cells, myeloid cells, and dendritic cells (DC) in the spleen of recipient mice 14 weeks after transplantation. n = 4–5. ( K and L ) Cell cycle analysis of DN3 thymocytes in 6-week-old control (Ctrl) and Sel1l CKO mice using Ki67 and DAPI. Representative flow cytometry plots ( K ) and quantification ( L ) are shown. n = 3. Data are shown as mean ± s.d. The statistical significance was calculated by two-tailed unpaired t-test ( C, E-J ) or Two-way ANOVA with Bonferroni test ( L ). n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure 2—figure supplement 2—source data 1. Excel file containing numerical values shown in .

Article Snippet: Other , CD4 (L3T4) MicroBeads, mouse 1 x 2 mL , 130-117-043 , Miltenyi Biotec , .

Techniques: Flow Cytometry, Control, Quantitative RT-PCR, Derivative Assay, Transplantation Assay, Cell Cycle Assay, Two Tailed Test

Journal: eLife

Article Title: Notch-induced endoplasmic reticulum-associated degradation governs mouse thymocyte β−selection

doi: 10.7554/eLife.69975

Figure Lengend Snippet: ( A and B ) Representative images of thymus ( A ) and quantification of total thymocytes, DP, SP4 and SP8 thymocytes ( B ) from age (6-week-old) and gender-matched control (Ctrl, Sel1l flox/flox ), Sel1l KO ( Sel1l flox/flox ; CD2 -iCre), Perk KO ( Perk flox/flox ; CD2 -iCre)and Sel1l / Perk double knockout (DKO. Sel1l flox/flox , Perk flox/flox ; CD2 -iCre) mice. n = 3–5 each group. ( C and D ) Representative images of spleen ( C ) and quantification of total splenocytes, total CD3 + T cells, CD4 + T cells, and CD8 + T cells ( D ) from the same mice with indicated genotype as in A and B . n = 3–5 each group. ( E and F ) Representative images of the inguinal (left) lymph node ( E ) and quantification of total lymphocytes, total CD3 + T cells, CD4 + T cells, and CD8 + T cells ( F ) from the same mice with indicated genotype as in A and B . n = 3–5 each group. ( G ) Quantitative RT–PCR analysis of Chop expression in DN3 thymocytes sorted from mice with indicated genotype. n = 3–5 each group. ( H and I ) Representative images ( H ) and quantification ( I ) of cleaved caspase-3 (CC3)-positive cells in the thymus of 6- to 8-week-old gender-matched mice with indicated genotype. Twelve fields were counted at ×20 magnification from four mice with indicated genotype. Scale bars are indicated. Data are representative of three independent experiments and are shown as mean ± s.d. The statistical significance was calculated by two-tailed unpaired t-test ( D, F ) One-way ANOVA with turkey test ( B, G ) or one-way ANOVA with Bonferroni test ( I ). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure 6—source data 1. Excel file containing numerical values shown in .

Article Snippet: Other , CD4 (L3T4) MicroBeads, mouse 1 x 2 mL , 130-117-043 , Miltenyi Biotec , .

Techniques: Control, Double Knockout, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: eLife

Article Title: Notch-induced endoplasmic reticulum-associated degradation governs mouse thymocyte β−selection

doi: 10.7554/eLife.69975

Figure Lengend Snippet:

Article Snippet: Other , CD4 (L3T4) MicroBeads, mouse 1 x 2 mL , 130-117-043 , Miltenyi Biotec , .

Techniques: Transgenic Assay, Sequencing, Mutagenesis, Recombinant, Reverse Transcription, SYBR Green Assay, In Situ, Staining, Live Cell Imaging, Synthesized, Software, Red Blood Cell Lysis

Journal: Nature Communications

Article Title: Defective autophagy in CD4 T cells drives liver fibrosis via type 3 inflammation

doi: 10.1038/s41467-025-59218-y

Figure Lengend Snippet: a GSEA from publicly available single-cell data of autophagy pathway genes in CD4 and CD8 T cells subsets from healthy and cirrhotic human livers ( n = 5 individuals/group) ( b ) Heatmap of pseudo-bulk autophagic gene expression from the leading edge of the GSEA graph of the CD4 T subset. c Representative histograms and Geometric Mean Fluorescence Intensity (geoMFI) of LC3II + CD4 T cells in human intrahepatic leukocytes from patients with extended fibrosis/cirrhosis (F3-F4, METAVIR score) ( n = 5) and controls ( n = 8), incubated with 30 μM chloroquine (CQ) or vehicle (# p = 0.0156 by two-tailed Wilcoxon matched-pairs signed rank test and * p = 0.0186 by two-tailed Mann–Whitney test). d Representative confocal immunofluorescent images and percentage of LC3 (green) colocalized with LAMP1 (red) over total LC3 in CD4 T cells from liver ( n = 9/group) and blood isolated from patients with extended fibrosis/cirrhosis ( n = 13) compared to controls ( n = 12) * p = 0.037. Scale bar is 1.99 μm. e Representative western blot analysis and quantification normalized to β-actin of autophagy-related proteins in CD4 T cells from blood of healthy and patients with mild or extended fibrosis/cirrhosis. Results are expressed as a fold over healthy except for pULK1 Ser 757 ( n = 12 healthy, n = 8 F1-F2, n = 16 F3-F4 and * p = 0.049 for Rubicon; n = 11 healthy, n = 8 F1-F2, n = 14 F3-F4 and ** p = 0.005 for P62; n = 15 healthy, n = 8 F1-F2, n = 10 F3-F4 for ATG5-ATG12; n = 9 healthy, n = 10 F3-F4 and * p = 0.026 for pULK Ser 757 ). (d-e) Data are mean ± S.E.M. Statistical analysis was performed by ( a ) GSEA ( d ) two-tailed Mann–Whitney for liver CD4 samples and ( d – e ) two-tailed univariate analysis for blood samples in which age of controls and patients differed statistically. Each variable achieving a p -value < 0.05 (i.e., only Rubicon) was then introduced into a two-tailed bivariate model (Table ). Source data are provided as a Source Data file.

Article Snippet: CD4 T cells were isolated from frozen PBMCs or IHLs using CD4 microbeads (Miltenyi Biotec, cat#130-045-101), according to manufacturer’s instructions.

Techniques: Gene Expression, Fluorescence, Incubation, Two Tailed Test, MANN-WHITNEY, Isolation, Western Blot

Journal: Nature Communications

Article Title: Defective autophagy in CD4 T cells drives liver fibrosis via type 3 inflammation

doi: 10.1038/s41467-025-59218-y

Figure Lengend Snippet: Mice were ( a – g ) injected CCl 4 for 5 weeks or ( h ) subjected to BDL. a , b Flow cytometry analysis of activation markers CD25 ( n = 7 ATG5 fl/fl and n = 6 ATG5 Tlymph-/- mice) and CD69 ( n = 8 ATG5 fl/fl and n = 4 ATG5 Tlymph-/- mice) among ( a ) CD4+ ** p = 0.0012 for CD25 and ** p = 0.0081 for CD69 and ( b ) CD8 + T cells. c – e RNA-seq analysis of purified CD4 + T cells isolated from CCl 4 -injected mice ( n = 5 ATG5 fl/fl and n = 6 ATG5 Tlymph-/- mice). c Volcano plot indicating number of significantly up- and down-regulated genes ( adj p ≤ 0.05). d Top KEGG pathways significantly enriched in ATG5 TLymph-/- compared to ATG5 fl/fl CD4 T cells. KEGG pathways are ordered by NES. e Selected genes significantly upregulated in ATG5 TLymph-/- compared to ATG5 fl/fl CD4 T cells. f Absolute numbers of non-pathogenic (IL-17A + IFN-γ- and IL−17A + IL-22 + , * p = 0.0478), pathogenic Th17 (IL-17A + IFN-γ+, * p = 0.0344), and GM-CSF+ among CD4 + T cells ( n = 7 mice/group) in CCl 4 injected mice. g – j Frequencies and dot plots of non-pathogenic and pathogenic Th17 among CD4 + T cells in ( g – h ) CCl 4 model ( n = 23 ATG5 fl/fl and n = 22 ATG5 Tlymph-/- mice, pooled data from 4 experiments, **** p < 0.0001 for IL-17A + IFN-γ- and IL-17A + IFN-γ+; n = 13 ATG5 fl/fl and n = 14 ATG5 Tlymph-/- mice, pooled data from 2 experiments, ** p = 0.0045 for IL-17A + IL-22+ and n = 7 mice/group, *** p = 0.0006 for GM-CSF + ) and (i-j) BDL model ( n = 10 ATG5 fl/fl and n = 9 ATG5 Tlymph-/- mice for IL-17A + IFN-γ-, IL-17A + IFN-γ + * p = 0.0289 and GM-CSF+ * p = 0.0295, p ooled data from 3 experiments and n = 6 mice/group for IL-17A + IL-22 + , pooled data from 2 experiments). a , b , f , g , i Data are mean ± S.E.M. Statistical analysis was performed by ( a , f , g , i ) two-tailed Mann–Whitney, ( c , e ) DESeq2 and p-values were adjusted using the Benjamini & Hochberg method ( d ) GSEA and p -values were adjusted using Benjamini-Hochberg method. Source data are provided as a Source Data file.

Article Snippet: CD4 T cells were isolated from frozen PBMCs or IHLs using CD4 microbeads (Miltenyi Biotec, cat#130-045-101), according to manufacturer’s instructions.

Techniques: Injection, Flow Cytometry, Activation Assay, RNA Sequencing, Purification, Isolation, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Defective autophagy in CD4 T cells drives liver fibrosis via type 3 inflammation

doi: 10.1038/s41467-025-59218-y

Figure Lengend Snippet: a Experimental co-culture protocol of MF with either non-activated or activated spleen ATG5 TLymph-/- or WT CD4 T cells ( b ) BrdU incorporation in hepatic MF following direct co-cultures with n = 4 non-activated or activated CD4 T cell preparations. c – e ELIZA quantification of cytokine/chemokine levels in supernatant from MF ( c ) cocultured with non-activated or activated CD4 T cells ( n = 8 CD4 + T cell isolations; n = 3 experiments for MF alone, * p = 0.0134, ** p = 0.0047 for CCL2, ** p = 0.0065, *** p = 0.0002 for IL-6 by two-tailed Mann–Whitney and ## p = 0.0078 by two-tailed Wilcoxon matched-pairs signed rank test for CCL2), ( d ) following direct contact or exposure to the conditioned medium (CM) of activated CD4 T cells ( n = 6 different CD4 T cell isolations, * p = 0.0411, ** p = 0.0022 by two-tailed Mann–Whitney) and ( e ) exposed to CM of activated ATG5 Tlymph-/- or WT CD4 T cells, following neutralization with 10 μg/ml IL-17A antibody or isotype ( n = 12 different CD4 T cell isolations for isotypes group and n = 10 for ATG5 TLymph-/- CD4+anti-IL-17A, ** p = 0.0029 for CCL2, * p = 0.0261, *** p = 0.0004 for CXCL10, *** p = 0.0004 for IL-6, **** p = < 0.0001for KC/IL-8 by two-tailed Mann–Whitney and ## p = 0.0059 for CCL2, ## p = 0.0098 for IL-6, ## p = 0.0020 for KC/IL-8 by two-tailed Wilcoxon matched-pairs signed rank test). Results are expressed as fold over MF +activated ATG5 fl/fl CD4 T cells. f Experimental co-culture protocol of primary hepatocytes with CM from activated CD4 T cells from either ATG5 TLymph-/- or WT mice. When indicated 10 μg/ml anti-IL-17A neutralizing antibody or control isotype was added. g mRNA gene expression of chemokines and cytokines in hepatocytes ( n = 9 different CD4 T cell isolations, ** p = 0.0019 for Cxcl1 , *** p = 0.0005 (ATG5 fl/fl CD4+isotype vs ATG5 TLymph-/- CD4+isotype), *** p = 0.0003 (ATG5 fl/fl CD4+isotype vs ATG5 TLymph-/- CD4+anti-IL-17A) for Cxcl10 , *** p = 0.0004 for Ccl2 , ** p = 0.0084 for Il6 by two-tailed Mann–Whitney and ## p = 0.0078 for Cxcl1 , # p = 0.0391 for Cxcl2 , # p = 0.0156 by two-tailed Wilcoxon matched-pairs signed rank test). Results are expressed as fold over hepatocytes +activated ATG5 fl/fl CD4 T cells. Data are mean ± S.E.M. ns: not significant. Source data are provided as a Source Data file. a , f Created in BioRender. Gilgenkrantz, H. (2025) https://BioRender.com/l88g711 .

Article Snippet: CD4 T cells were isolated from frozen PBMCs or IHLs using CD4 microbeads (Miltenyi Biotec, cat#130-045-101), according to manufacturer’s instructions.

Techniques: Co-Culture Assay, BrdU Incorporation Assay, Two Tailed Test, MANN-WHITNEY, Neutralization, Control, Gene Expression

Journal: Nature Communications

Article Title: Defective autophagy in CD4 T cells drives liver fibrosis via type 3 inflammation

doi: 10.1038/s41467-025-59218-y

Figure Lengend Snippet: a Flow cytometry analysis of the frequency and cytokine production of intrahepatic CD11b + F4/80+ MoMac from CCl 4 -exposed ATG5 TLymph-/- and ATG5 fl/fl mice ( n = 13 ATG5 fl/fl and n = 12 ATG5 TLymph-/- mice, pooled data from 2 experiments, * p = 0.0257 for IL-1α, * p = 0.0457 for pro-IL-1β). b Experimental protocol of bone marrow-derived macrophages (BMDMs)/CD4 T cell co-culture created in BioRender. Gilgenkrantz, H. (2025) https://BioRender.com/l88g711 . c (left) Intracellular staining of 1 ng/ml LPS-stimulated BMDMs for IL-1α and pro-IL-1β. (right) ELIZA on the co-culture supernatant ( n = 8 different isolations, * p = 0.0145, ** p = 0.0045, *** p = 0.002). d Representative dot plots and mean quantification of IL-1α and pro-IL-1β in BMDMs co-cultures ( n = 4 mice/group), transwell ( n = 4 mice/group, * p = 0.0286), or in the presence of 10 μg/ml control isotype, IL-17A ( n = 4 mice/group, * p = 0.0286)or GM-CSF neutralizing antibody for 24 hrs ( n = 8 mice/group, ** p = 0.0028, * p = 0.0390 and ## p = 0.0078 by two-tailed Wilcoxon matched-pairs signed rank test). Data are mean ± S.E.M. Statistical analysis was performed by two-tailed Mann-Whitney, unless otherwise indicated. Source data are provided as a Source Data file.

Article Snippet: CD4 T cells were isolated from frozen PBMCs or IHLs using CD4 microbeads (Miltenyi Biotec, cat#130-045-101), according to manufacturer’s instructions.

Techniques: Flow Cytometry, Derivative Assay, Co-Culture Assay, Staining, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Defective autophagy in CD4 T cells drives liver fibrosis via type 3 inflammation

doi: 10.1038/s41467-025-59218-y

Figure Lengend Snippet: a Western blot analysis of CD4 T cell lysates from patients with extended fibrosis/cirrhosis exposed to 1 µM AZD8055 or vehicle for 3 hrs. LC3 expression was measured in the presence of 30 µM CQ ( n = 7 patients, # p = 0.0156). Lanes have been rearranged; non-adjacent lanes are separated by a vertical black line. b Flow cytometry analysis of the frequency of IL-17A + IFN-γ + and IFN-γ + cells among CD4 + T cells ( n = 8 patients, ## p = 0.0078, # p = 0.0312) and ELIZA quantification of GM-CSF secretion in PBMCs from patients with extended fibrosis/cirrhosis exposed to 10 μM AZD8055 ( n = 7 patients). c , d RUBCN Tlymph-/- and their WT counterparts mice were injected with CCl 4 twice a week for 5 weeks. c Representative images and quantification of SR ( n = 18 RUBCN fl/fl and n = 14 RUBCN TLymph-/- mice, pooled data from 4 experiments, ** p = 0.0065) and α-SMA + areas in liver tissue sections ( n = 10 mice/group, pooled data from 2 experiments, *** p = 0.0006). d Hepatic expression of inflammatory cytokines, chemokines, and chemokine receptors genes ( n = 18 RUBCN fl/fl and n = 14 RUBCN TLymph-/- mice, pooled data from 4 experiments, * p = 0.0282 for Il1b , * p = 0.0205 for Tnfa , * p = 0.0139 for Ccl2 and * p = 0.040 for Cxcl10 ). e Flow cytometry analysis of IL-17A + IFN-γ-, IL-17A + IFN-γ + ( n = 17 mice/group, pooled data from 3 experiments), and IL-17A + IL-22+ ( n = 12 RUBCN fl/fl and n = 14 RUBCN TLymph-/- mice, pooled data from 2 experiments, * p = 0.0322) CD4 T cell frequencies in the liver of RUBCN TLymph-/- and WT littermates exposed to CCl 4 . Scale bar is 100 μm. b – e Data are mean ± S.E.M. Statistical analysis was performed by ( a , b ) two-tailed Wilcoxon matched-pairs signed rank and ( c – e ) by two-tailed Mann-Whitney test. Source data are provided as a Source Data file.

Article Snippet: CD4 T cells were isolated from frozen PBMCs or IHLs using CD4 microbeads (Miltenyi Biotec, cat#130-045-101), according to manufacturer’s instructions.

Techniques: Western Blot, Expressing, Flow Cytometry, Injection, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Defined metabolic states shape T cell fate and function across culture conditions

doi: 10.3389/fimmu.2025.1703095

Figure Lengend Snippet: Multiparametric assessment identifies variation in T-cell activation across conditions. (A) Schematic of the experimental workflow and metabolic pathways assessed. CD3 + T cells from 2–3 donors were stimulated under four defined activation/media conditions (TexMACS [TM] or ICXF media combined with ImmunoCult [SC] or TransAct [TA] CD3/CD28 activators) and profiled on Day 3 for activation markers, cytokine secretion, and metabolic features. (B–K) Comparison of not activated (Not Act) and activated (Act) T cells. (B) Average cell volume (fL). (C) Percentage of CD25 + or CD69 + T cells measured by flow cytometry. (D) Cytokine secretion (IFNγ, TNFα, IL-2) measured 48 h post-activation. Not Act samples, which showed minimal or no cytokine production, are grouped together for display. Statistical comparisons were made between each Act condition and the Not Act group for each cytokine. (E) Metabolic reducing potential (measured as relative luminescence units, RLU) over 72 h for three conditions: Not Act, TM + SC, and ICXF + TA. (F–H) Intracellular ATP, NAD+, and total NADP + NADPH levels (all in fmol/cell). (I, J) Glucose consumption and lactate secretion (both in pmol/hr/cell) measured in culture media. (K) Malate accumulation levels (fmol/hr/cell). Each graph includes data from 2–3 donors. Shapes represent stimulation conditions. Each data point represents the average of 2–3 biological replicates (independent cultures) for a given donor and condition. Statistical comparisons used two-tailed, unpaired Mann–Whitney tests. Significance levels: p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).

Article Snippet: Cells were activated using either T cell TransAct (Miltenyi Biotec, Cat. #130-111-160) or ImmunoCult Human CD3/CD28 T Cell Activator (STEMCELL Technologies, Cat. #10971) and supplemented with IL-7 and IL-15 each at 2.5 ng/mL (Miltenyi Biotec, Cat. #130-095–361 and #130-095-764).

Techniques: Activation Assay, Comparison, Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Defined metabolic states shape T cell fate and function across culture conditions

doi: 10.3389/fimmu.2025.1703095

Figure Lengend Snippet: Activation medium drives metabolic profile of T cells. (A) Schematic of the experimental workflow and metabolic pathways investigated. T cells from a single donor were activated under 48 conditions combining three media types, four CD3/CD28-based activators, and two cytokine cocktails at two concentrations. Cells were harvested on Day 3 for metabolic profiling. (B, C) Principal component analysis (PCA) of intracellular metabolite profiles. (B) PCA colored by medium and marked by activator to visualize condition-dependent variation. (C) PCA colored by K-means cluster (k = 4), identifying four metabolically distinct groups. (D–J) Metabolite levels grouped by K-means cluster. (D) Metabolic reducing potential (measured as relative luminescence units, RLU). (E–G) Intracellular metabolite levels: ATP (µM), NAD+ (nM), and total NADP(H) (RLU). (H, I) Extracellular metabolite concentrations: glucose consumption and lactate secretion (both in mM). (J) Malate levels (µM). Each point represents the average of four technical replicates for a given activation condition. Box plots represent Tukey distribution: boxes span the interquartile range (IQR), whiskers extend to 1.5× IQR, and diamonds indicate outliers. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. Significance levels are indicated as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Article Snippet: Cells were activated using either T cell TransAct (Miltenyi Biotec, Cat. #130-111-160) or ImmunoCult Human CD3/CD28 T Cell Activator (STEMCELL Technologies, Cat. #10971) and supplemented with IL-7 and IL-15 each at 2.5 ng/mL (Miltenyi Biotec, Cat. #130-095–361 and #130-095-764).

Techniques: Activation Assay, Metabolic Labelling

Journal: Frontiers in Immunology

Article Title: Defined metabolic states shape T cell fate and function across culture conditions

doi: 10.3389/fimmu.2025.1703095

Figure Lengend Snippet: Metabolic signatures predict expansion outcomes following activation. (A) Schematic of the expansion workflow. T cells from 2–3 donors were activated under eight representative conditions ( Table 1 ; abbreviations as defined there) and transferred to GREX24 vessels on Day 3 for continued culture. (B, C) Metabolic ratios calculated from the rate of metabolite secretion or consumption between Day 3 and Day 7: lactate-to-glucose ratio (B) and lactate-to-malate ratio (C) . (D) Fold expansion from Day 3 to Day 7 is shown as box plots (left axis). Viability on Day 7 is overlaid as pink dots (right axis). (E) Correlation between lactate levels on Day 3 (mM) and fold expansion through Day 7. Pearson correlation: r = 0.6840, p = 3.079e–07. Box plots (B–D) show Tukey distribution. Statistical comparisons were performed for panels B–C using one-way ANOVA followed by Tukey’s multiple comparisons test. For panel C, all pairwise comparisons were performed, but significance is summarized using a single bracket to indicate the overall difference between TexMACS and ICXF conditions for clarity. Significance levels are indicated as follows: p < 0.05 (*), p < 0.01 (**), and p < 0.0001 (****).

Article Snippet: Cells were activated using either T cell TransAct (Miltenyi Biotec, Cat. #130-111-160) or ImmunoCult Human CD3/CD28 T Cell Activator (STEMCELL Technologies, Cat. #10971) and supplemented with IL-7 and IL-15 each at 2.5 ng/mL (Miltenyi Biotec, Cat. #130-095–361 and #130-095-764).

Techniques: Activation Assay

Journal: Frontiers in Immunology

Article Title: Defined metabolic states shape T cell fate and function across culture conditions

doi: 10.3389/fimmu.2025.1703095

Figure Lengend Snippet: Targeted inhibition confirms media-dependent metabolic differences in T cells. (A–D) Percent inhibition of metabolic and expansion metrics following treatment with 2DG, Antimycin A, or Rotenone, relative to untreated controls in matched conditions. Data are shown for TexMACS (blue) and ImmunoCult-XF (orange) media. (A, B) Intracellular ATP (A) and NAD (B) levels measured on Day 3. (C) Percent inhibition of lactate secretion rate from Day 3 to Day 7. (D) Growth rate inhibition calculated from fold expansion between Day 3 and Day 7. Box plots represent Tukey distribution. Data summarize two donors. Statistical comparisons were performed using Welch’s unpaired t-tests. Significance levels are indicated as follows: ns=not significant, p < 0.05 (*), p < 0.01 (**), and p < 0.0001 (****).

Article Snippet: Cells were activated using either T cell TransAct (Miltenyi Biotec, Cat. #130-111-160) or ImmunoCult Human CD3/CD28 T Cell Activator (STEMCELL Technologies, Cat. #10971) and supplemented with IL-7 and IL-15 each at 2.5 ng/mL (Miltenyi Biotec, Cat. #130-095–361 and #130-095-764).

Techniques: Inhibition

Journal: Frontiers in Immunology

Article Title: Defined metabolic states shape T cell fate and function across culture conditions

doi: 10.3389/fimmu.2025.1703095

Figure Lengend Snippet: Functional responses of T cells activated in different media conditions. (A–C) Functional assessments were performed on Day 10 post-activation using T cells expanded under representative media and activation conditions. (A) Memory phenotype distribution of CD4 + and CD8 + T cell subsets assessed by flow cytometry using CD45RA and CD62L expression. Box plots represent Tukey distribution. (B) Specific lysis (%) of HiBiT Ramos target cells co-cultured with T cells and blinatumomab at seven effector-to-target (E:T) ratios. Lysis was calculated relative to spontaneous release (Ramos cells alone, no T cells) and maximum lysis (digitonin-treated Ramos). (C) Quantification of cytotoxic activity based on area under the curve (AUC) from E:T response curves. Data summarize two donors. Statistical comparisons were performed using one-way ANOVA with Tukey’s multiple comparisons test. Significance levels are indicated as follows: ns=not significant, p < 0.05 (*), p < 0.01 (**), and p < 0.0001 (****). TM, TexMACS; TA, TransAct; SC, StemCell ImmunoCult.

Article Snippet: Cells were activated using either T cell TransAct (Miltenyi Biotec, Cat. #130-111-160) or ImmunoCult Human CD3/CD28 T Cell Activator (STEMCELL Technologies, Cat. #10971) and supplemented with IL-7 and IL-15 each at 2.5 ng/mL (Miltenyi Biotec, Cat. #130-095–361 and #130-095-764).

Techniques: Functional Assay, Activation Assay, Flow Cytometry, Expressing, Lysis, Cell Culture, Activity Assay

Journal: Oncoimmunology

Article Title: A liposomal RNA vaccine inducing neoantigen-specific CD4 + T cells augments the antitumor activity of local radiotherapy in mice

doi: 10.1080/2162402X.2020.1771925

Figure Lengend Snippet: A CD4 neoantigen vaccine improves LRT-mediated survival of mice with CT26 tumors in a CD8 + T cell-dependent manner. (a–c) CT26 tumor growth (a) and survival (b) of BALB/c mice (n = 7–8/group) locally irradiated with 12 Gy or 3 × 6 Gy at a mean volume of 60 mm 3 . (c) gp70-AH1 tetramer + CD8 + T cells in blood of treated mice (n = 4–5/group). (d-f) CT26 tumor growth (d) and survival (e) of mice (n = 7–12/group) locally irradiated with 12 Gy at a mean tumor volume of ~70 mm 3 and immunized three times with CT26 P ME 1 or control RNA-LPX. (f) Gp70-AH1 tetramer + CD8 + T cells in blood of treated mice (n = all mice/group). (g-i) CT26 tumor growth (g) and survival (h) of mice (n = 8–9/group) immunized with CT26 P ME 1 or control RNA and locally irradiated at a mean tumor volume of 90 mm 3 . CD8 + T cells were depleted 6 days after LRT, administering the anti-CD8 antibody every 3–4 days over 3 weeks. (i) Gp70-AH1 tetramer + CD8 + T cells in blood of treated mice (n = 8–9/group). Significance was determined using (b, e, h) Mantel-cox log-rank test and (c, f, i) one-way ANOVA, Tukey’s multiple comparison test. (a, d, g) Tumor growth is displayed on a log 2 -scale. Ratios depict frequency of mice with complete tumor responses (CR). Mean±SEM. nd = not determined.

Article Snippet: For the isolation of tumor CD45 + or CD8 + cells, target cells were magnetically enriched using the mouse CD8 (TIL) or CD45 (TIL) MicroBeads and for spleen CD4 + or CD8 + T cells using the CD4 (L3T4) or CD8a (Ly-2) MicroBeads (Miltenyi Biotech) according to manufacturer’s instructions.

Techniques: Irradiation, Control, Comparison

Journal: Oncoimmunology

Article Title: A liposomal RNA vaccine inducing neoantigen-specific CD4 + T cells augments the antitumor activity of local radiotherapy in mice

doi: 10.1080/2162402X.2020.1771925

Figure Lengend Snippet: Activated poly-functional Th1-like CD4 + T cells against the immunodominant vaccine-encoded CD4 neoantigen are induced in the spleen of CD4 neoantigen vaccine/LRT treated mice. (a–c) CT26 tumor growth (a) and survival (b) of BALB/c mice (n = 7–10/group) treated with 12 Gy at a mean tumor volume of 45 mm 3 and immunized with different RNA-LPX vaccines. RNA-LPX vaccines included CT26 P ME 1; CT26 ME1, the most immunogenic CT26 P ME 1 neoantigen; P ctrl. encoding mutations not expressed in CT26 and control RNA-LPX, encoding no antigens at all. (c) Gp70-AH1 tetramer + CD8 + T cells in treated mice (n = 7–10/group). (d, e) Phenotypic analysis of enriched splenic CD4 + T cells from mice immunized with CT26 P ME 1 or control RNA-LPX (n = 5/group). (d) Differential expression of ICOS, CD69, PD-1, CD62L, T-bet, CD25 on ME1-specific (ME1tet + ) and -nonspecific (ME1tet − ) CD4 + T cells as determined by flow cytometry. Representative pseudocolor plots of ME1 tetramer staining are shown. (e) Supernatant cytokine secretion after 48 h co-culture of CD4 + T cells with ME1-peptide-loaded BALB/c BMDC. (f) IFNγ intracellular cytokine staining after ex vivo re-stimulation of enriched splenic CD4 + T cells from CT26-tumor-bearing mice, locally 12 Gy irradiated at a mean tumor volume of 60 mm 3 and immunized with CT26 P ME 1 or control RNA-LPX, with ME1 peptide-loaded BMDC (n = 6/group). Significance was determined using (b) Mantel-Cox log-rank test, (c, d, f) one-way ANOVA, Tukey’s multiple comparison test and (e) unpaired, two-tailed Student’s t-test. (a) Tumor growth is displayed on a log 2 -scale. Ratios depict frequency of mice with complete tumor responses (CR). Mean±SEM.

Article Snippet: For the isolation of tumor CD45 + or CD8 + cells, target cells were magnetically enriched using the mouse CD8 (TIL) or CD45 (TIL) MicroBeads and for spleen CD4 + or CD8 + T cells using the CD4 (L3T4) or CD8a (Ly-2) MicroBeads (Miltenyi Biotech) according to manufacturer’s instructions.

Techniques: Functional Assay, Vaccines, Control, Quantitative Proteomics, Flow Cytometry, Staining, Co-Culture Assay, Ex Vivo, Irradiation, Comparison, Two Tailed Test

Journal: Oncoimmunology

Article Title: A liposomal RNA vaccine inducing neoantigen-specific CD4 + T cells augments the antitumor activity of local radiotherapy in mice

doi: 10.1080/2162402X.2020.1771925

Figure Lengend Snippet: Adding the CD4 neoantigen vaccine to LRT results in a potent polyantigenic CD8 + T cell response and T cell memory. (a) IFNγ ELISpot using splenocytes isolated from CT26 tumor-bearing BALB/c mice, vaccinated with CT26 P ME 1 or control RNA-LPX and locally irradiated at a mean tumor volume of 45 mm 3 (n = 6 mice/group, 2 mice pooled each), against CT26 cells, gp70-AH1 peptide-pulsed BALB/c BMDC, CT26-gp70KO, and 20 Gy irradiated CT26-gp70KO cells. In vitro irradiation was performed to enhance tumor cell MHC class I presentation (Supplementary Figure 3(a)). (b-d) CT26-gp70KO tumor growth (b) and survival (c) of mice immunized with CT26 P ME 1 or control RNA-LPX and irradiated at a mean volume of 45 mm 3 (n = 7–10/group). (d) IFNγ ELISpot using splenocytes isolated from CT26 tumor-bearing mice (n = 4 mice/group, 2 mice pooled each) against CT26 cells, 20 Gy irradiated CT26-gp70KO cells and gp70-AH1 peptide-pulsed BALB/c BMDC. As in (a), in vitro irradiation was performed to enhance tumor cell MHC class I presentation (Supplementary Figure 3(a)). (e) Survival of 12 Gy and CT26 P ME 1/ME1 RNA-LPX treated CT26 tumor-free mice, challenged with a tumorigenic dose of CT26-gp70KO cells 40 days after initial tumor rejection (n = 10 each). Naïve BALB/c mice served as control group (n = 10). Significance was determined using (c, e) log-rank test and (a, d) one-way ANOVA, Tukey’s multiple comparison test. (b) Tumor growth is displayed on a log 2 -scale. Ratios depict frequency of mice with complete tumor responses (CR). Mean±SEM.

Article Snippet: For the isolation of tumor CD45 + or CD8 + cells, target cells were magnetically enriched using the mouse CD8 (TIL) or CD45 (TIL) MicroBeads and for spleen CD4 + or CD8 + T cells using the CD4 (L3T4) or CD8a (Ly-2) MicroBeads (Miltenyi Biotech) according to manufacturer’s instructions.

Techniques: Enzyme-linked Immunospot, Isolation, Control, Irradiation, In Vitro, Comparison

Journal: Oncoimmunology

Article Title: A liposomal RNA vaccine inducing neoantigen-specific CD4 + T cells augments the antitumor activity of local radiotherapy in mice

doi: 10.1080/2162402X.2020.1771925

Figure Lengend Snippet: Tumor immune infiltrates of CD4 neoantigen vaccine/LRT-treated mice consist of activated, tumor antigen-specific CD8 + T cells and vaccine-induced CD4 + T cells capable of providing cognate help. (a, b) Analysis of TIL in CT26 tumor-bearing BALB/c mice (n = 5–6/group) treated with 12 Gy at a mean tumor volume of 45 mm 3 and immunized with CT26 P ME 1 or control RNA-LPX. Tumors were resected 8 days after LRT. (a) Percentage of CD45 + , tumor cells, CD4 + and CD8 + T cells and (b) gp70-AH1 tetramer + CD8 + T cells. Representative pseudocolor plots show gp70-AH1 tetramer staining. (c-e) Analysis of tumor-infiltrating CD8 + T cells (c, d) and CD4 + T cells (e) from CT26 tumor-bearing mice locally irradiated with 12 Gy at a mean tumor volume of 60 mm 3 and immunized three times with CT26 P ME 1 or control RNA-LPX (n = 6/group). CD4 + and CD8 + T cells were enriched via CD45 + TIL MACS for (c) phenotypic analysis or (d, e) intracellular cytokine staining after restimulation with gp70-AH1 or ME1 peptide-loaded BALB/c BMDC. Representative pseudocolor plots show CD8 + (d, right) and CD4 + (e, right) T cells after peptide restimulation. (f, g) TCRβ CDR3 sequencing of tumor CD8 + T cells from mice (n = 3–4/group), irradiated with 12 Gy at a mean tumor volume of ~100 mm 3 and immunized three times with CT26 P ME 1 RNA-LPX or control RNA-LPX, analyzed 9 days after irradiation. (g) Number of unique TCRβ sequences in tumors or treated mice (left) and mean TCRβ clonotype frequency (clonality) of CD8 + T cells (right). (f) Frequency of blood-tumor shared TCRβ clones prior and 9 days after LRT compared to all unique tumor clones found 9 days after LRT. Significance was determined using (a–g) one-way ANOVA, Tukey’s multiple comparison test. Mean±SEM.

Article Snippet: For the isolation of tumor CD45 + or CD8 + cells, target cells were magnetically enriched using the mouse CD8 (TIL) or CD45 (TIL) MicroBeads and for spleen CD4 + or CD8 + T cells using the CD4 (L3T4) or CD8a (Ly-2) MicroBeads (Miltenyi Biotech) according to manufacturer’s instructions.

Techniques: Control, Staining, Irradiation, Sequencing, Clone Assay, Comparison

Journal: Molecular Cancer

Article Title: Intracellular osteopontin protects from autoimmunity-driven lymphoma development inhibiting TLR9-MYD88-STAT3 signaling

doi: 10.1186/s12943-022-01687-6

Figure Lengend Snippet: Characterization of lymphomas occurring in Fas lpr/lpr and OPN-/-Fas lpr/lpr mice. A . Graph showing the incidence of splenic lymphoma in Fas lpr/lpr ( n = 46) and OPN-/-Fas lpr/lpr ( n = 48) mice at about 5–6 months of age (69% and 33% and respectively) (****, p < 0.0001; Chi-square two-tailed test). B . Representative H&E staining of Fas lpr/lpr and OPN-/-Fas lpr/lpr tumours. Scale bar: 50 μm. C . An example of flow cytometry analysis showing the expression of surface IgM and B220 on CD19 + B cells from naïve and autoimmune mice. D . IF for BCL6 (upper panel) and Ki67 (lower panel) on splenic lymphomas from Fas lpr/lpr and OPN-/-Fas lpr/lpr mice. PAX5 is used as pan B cell marker. Scale bar: 100 μm. E . Flow cytometry quantification of CD19 + Ki67 + cells in the spleen from Fas lpr/lpr ( n = 7) and OPN-/-Fas lpr/lpr animals ( n = 7) (Student t test; *, p: 0.0153). The graph shows a pool of two independent experiments. F . Western blot illustrating the expression of BCL2, c-MYC and IRF4, in splenic CD19 + B cells from Fas lpr/lpr ( n = 3) and OPN-/-Fas lpr/lpr ( n = 3) mice. CD19 + cells from a BALB/c and a OPN-/- mouse were used as controls. G . Quantification of western blot, relative to β-ACTIN as housekeeping gene, of BCL2 (Student t test; **, p: 0.0076), c-MYC (Student t test; ns, p: 0.4781) and IRF4 (Student t test; *, p : 0.0260). Statistics was applied to values related to Fas lpr/lpr and OPN-/-Fas lpr/lpr B cells

Article Snippet: CD19 + cells were isolated from mouse spleens using Microbeads anti-CD19 + (Miltenyi, #130–121-301) and the purity of cells was assessed by flow cytometry using an antibody against CD19 (CD19 FITC (BD, clone CL1D3, #557,398).

Techniques: Two Tailed Test, Staining, Flow Cytometry, Expressing, Marker, Western Blot

Journal: Molecular Cancer

Article Title: Intracellular osteopontin protects from autoimmunity-driven lymphoma development inhibiting TLR9-MYD88-STAT3 signaling

doi: 10.1186/s12943-022-01687-6

Figure Lengend Snippet: Evaluation of STAT3 pathway activation in CD19 + B cells from autoimmune mice. A . Western blot illustrating the expression of phospho- and total STAT3 in splenic CD19 + B cells from Fas lpr/lpr ( n = 3) and OPN-/-Fas lpr/lpr ( n = 3) mice. CD19 + from a BALB/c and a OPN-/- mouse were used as controls. B . Quantification of western blot, relative to β-ACTIN as housekeeping gene of ph-STAT3 (Student t test; ***, p:0.0007) and total STAT3 (ns, p: 0.1237). Statistics was applied to values related to Fas lpr/lpr and OPN-/-Fas lpr/lpr B cells. C . Representative IHC (upper panels) showing the expression of ph-STAT3 in OPN-sufficient and -deficient lymphomas. Scale bar: 100 μm. Representative IF (lower panels) for PAX5 (pan B cell marker) and ph-STAT3 on OPN + / + and -/-tumours. Scale bar: 50 μm. D . RT-PCR on purified splenic CD19 + cells from Fas lpr/lpr and OPN-/-Fas lpr/lpr mice showing the expression of STAT3- target genes Prdm1 ( Student t test; *, p: 0.0261 ) and Birc5 ( Student t test; *, p : 0.0195 ) . CD19 + from B/c and OPN-/- mice were used as controls. Values are shown as 2^-DCT. Data are a pool of two independent experiments with 3 samples for each group. E . Representative RNA scope imaging showing Prdm1 expression on spleens from autoimmune mice

Article Snippet: CD19 + cells were isolated from mouse spleens using Microbeads anti-CD19 + (Miltenyi, #130–121-301) and the purity of cells was assessed by flow cytometry using an antibody against CD19 (CD19 FITC (BD, clone CL1D3, #557,398).

Techniques: Activation Assay, Western Blot, Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Purification, RNAscope, Imaging

Journal: Molecular Cancer

Article Title: Intracellular osteopontin protects from autoimmunity-driven lymphoma development inhibiting TLR9-MYD88-STAT3 signaling

doi: 10.1186/s12943-022-01687-6

Figure Lengend Snippet: Gene expression profile of CD19 + cells from OPN-deficient and –sufficient animals. A . Volcano plot showing the up- and down-regulated genes comparing splenic CD19 + cells from OPN-/-Fas lpr/lpr and Fas lpr/lpr mice. B . PathfindR analysis on Reactome pathway illustrating the modulated signaling pathways in the comparison between OPN-/-Fas lpr/lpr and Fas lpr/lpr B cells. C . GSEA functional annotation of OPN-/-Fas lpr/lpr vs Fas lpr/lpr B cells gene expression data

Article Snippet: CD19 + cells were isolated from mouse spleens using Microbeads anti-CD19 + (Miltenyi, #130–121-301) and the purity of cells was assessed by flow cytometry using an antibody against CD19 (CD19 FITC (BD, clone CL1D3, #557,398).

Techniques: Gene Expression, Protein-Protein interactions, Comparison, Functional Assay

Journal: Molecular Cancer

Article Title: Intracellular osteopontin protects from autoimmunity-driven lymphoma development inhibiting TLR9-MYD88-STAT3 signaling

doi: 10.1186/s12943-022-01687-6

Figure Lengend Snippet: Expression of OPN and evaluation of TLR9-MYD88 signaling pathway in CD19 + cells from autoimmune mice . A . IHC for OPN performed on splenic tissues from BALB/c and autoimmune mice with and without lymphomas. Scale bar: 100 μm (left) and 50 μm (right). B . Representative double IF for OPN (red) and the endosomal marker CD63 (blu) on purified CD19 + cells from BALB/c and Fas lpr/lpr mice. Scale bar: 15 μm. C. Flow cytometry analysis showing CD86 expression on CD19 + cells from naïve and autoimmune mice ( n = 3) with or without 3-day stimulation with CpG 1826 (*, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001, Two-way ANOVA). D . Representative western blot showing the level of MYD88, IRAK4 and IRAK1 in CD19 + B cells purified from Fas lpr/lpr and OPN-/-Fas lpr/lpr mice at steady state condition and after TLR9-triggering with CpG 1826. The TLR4 agonist LPS and the TLR3 ligand Poly I:C were used as positive and negative controls, respectively. E . Western blot quantification relative to β-ACTIN housekeeping gene of MYD88, IRAK4 and IRAK1. F . Flow cytometry analysis showing the percentage of CD19 + TLR9 + cells (upper panel) and TLR9 protein level (MFI) on CD19 + cells (lower panel) from the spleen of Fas lpr/lpr ( n = 6) and OPN-/-Fas. lpr/lpr mice ( n = 6). B/c and OPN-/-mice were used as controls. Data in the graphs are referred to a pool of two independent experiments. (**, p < 0.01; Ordinary one way ANOVA) (*, p < 0.05; Ordinary one way ANOVA)

Article Snippet: CD19 + cells were isolated from mouse spleens using Microbeads anti-CD19 + (Miltenyi, #130–121-301) and the purity of cells was assessed by flow cytometry using an antibody against CD19 (CD19 FITC (BD, clone CL1D3, #557,398).

Techniques: Expressing, Marker, Purification, Flow Cytometry, Western Blot

Journal: Molecular Cancer

Article Title: Intracellular osteopontin protects from autoimmunity-driven lymphoma development inhibiting TLR9-MYD88-STAT3 signaling

doi: 10.1186/s12943-022-01687-6

Figure Lengend Snippet: Over-expression of sOPN and iOPN in OPN-/-Fas lpr/lpr DLBCL cell lines and evaluation of TLR9-MYD88 cascade. A . H&E (upper panels) and IF for PAX5/BCL6 (middle panels) performed on OPN-/-Fas lpr/lpr mouse spleens from which OPL239 and OPL241 derive. H&E staining on tumorigenic spleens from nu/nu mice intravenously injected with OPL239 and OPL241 (lower panels). Scale bar: 100 μm. B . RT-PCR showing the expression of Bcl6 , Bcl2 , Irf4 and c-myc in OPL239 and OPL241 cell lines. CD19 + from BALB/c and OPN-/-mice were used as controls. Values are shown as 2^-DCT. C . Western blot on OPL239 and OPL241 lysates illustrating the level of BCL2, c-MYC, IRF4 and the basal level/activation of STAT3 and NF-κB (p65) pathways. D . Western blot for phospho- and total STAT3, phospho- and total p65 (NF-κB) performed on OPL239, OPL239 IRES-Green, OPL239 Spp1-IRES-Green and OPL239 iOPN-IRES-Green with or without 30-min stimulation with CpG 1826. E . RT-PCR for Il6 and Tnfa on OPL239 cell variants with or without 3 h-stimulation with CpG 1826. Values are shown as 2^-DCT. (*, p < 0.05; Two-way ANOVA), (**, p < 0.01; Two-way ANOVA), (***, p < 0.001; Two-way ANOVA). F . Western blot on OPL241 variants illustrating the expression of phospho- and total STAT3 and NF-κB with or without 30-min stimulation with CpG 1826. G. RT-PCR for Il6 and Tnfa on OPL241 cell variants with or without 3 h-stimulation with CpG 1826. Values are shown as 2^-DCT. (*, p < 0.05; Two-way ANOVA), (**, p < 0.01, Two-way ANOVA)

Article Snippet: CD19 + cells were isolated from mouse spleens using Microbeads anti-CD19 + (Miltenyi, #130–121-301) and the purity of cells was assessed by flow cytometry using an antibody against CD19 (CD19 FITC (BD, clone CL1D3, #557,398).

Techniques: Over Expression, Staining, Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Activation Assay

Journal: Molecular Cancer

Article Title: Intracellular osteopontin protects from autoimmunity-driven lymphoma development inhibiting TLR9-MYD88-STAT3 signaling

doi: 10.1186/s12943-022-01687-6

Figure Lengend Snippet: In vivo effects of over-expression of the soluble and the intracellular OPN in OPN-/-Fas lpr/lpr DLBCL cells. A . Flow cytometry analysis showing the tumour take (% of CD19 + GFP +) in spleen and liver of 5 Gy irradiated BALB/c mice injected with OPL239 IRES-Green, OPL239 Spp1-IRES-Green and OPL239 iOPN-IRES-Green. Four mice per group were used in the experiment (*, p < 0.05; ****, p < 0.0001; Ordinary one way ANOVA). B . Flow cytometry analysis showing the percentage of lymphoid and myeloid populations in the spleen of mice injected with OPL239 cell variants [(T conv: T conventional cells; T reg: T regulatory cells) (*, p < 0.05, **, p < 0.001; ****, p < 0.0001; Ordinary one way ANOVA)]. An irradiated B/c mouse was used as control

Article Snippet: CD19 + cells were isolated from mouse spleens using Microbeads anti-CD19 + (Miltenyi, #130–121-301) and the purity of cells was assessed by flow cytometry using an antibody against CD19 (CD19 FITC (BD, clone CL1D3, #557,398).

Techniques: In Vivo, Over Expression, Flow Cytometry, Irradiation, Injection, Control

Journal: Molecular Neurodegeneration

Article Title: Early lysosomal maturation deficits in microglia triggers enhanced lysosomal activity in other brain cells of progranulin knockout mice

doi: 10.1186/s13024-018-0281-5

Figure Lengend Snippet: Cathepsin maturation is selectively impaired in Grn −/− microglia. a Schematic representation of the brain cell isolation using MACS Technology (Miltenyi Biotec) b PGRN expression in acutely isolated microglia, astrocytes and neurons enriched fractions of 4- month-old wt mice detected by immunoblotting. The identity of neural cell types was verified by detection of Iba1 for microglia, GFAP for astrocytes and Tuj1 for neurons. c-i Cathepsin expression and maturation in the CD11b-positive, microglia enriched, fraction and the CD11b-negative, microglia depleted cellular fraction isolated form cortices of brain from 3-month-old Grn +/+ (wt) and Grn −/− (ko) mice. Representative immunoblots for the cathepsin expression of CatD ( c, d ), CatB ( e, f ), CatL ( g, h ) and for CatS ( i ) (only microglia enriched fraction). The molecular weight standards in kilo Daltons (kDa) are indicated on the left side of all immunoblots. A dotted line in the blot indicates that samples of heterozygous mice were cut out, but all samples were loaded on one gel. Quantification of immunoblots for total cathepsin or maturation variants pro-form (p), single chain (sc) and heavy chain (hc) normalized to wt are shown as mean ± SD. For statistical analysis the unpaired, two-tailed student’s t-test was used to compare ko to wt mice (n = 3–5) (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant) ( c-i )

Article Snippet: CD11b-positive microglia were magnetically labelled with CD11b MicroBeads, loaded onto a MACS Column (Miltenyi Biotec) and subjected to magnetic separation.

Techniques: Cell Isolation, Expressing, Isolation, Western Blot, Molecular Weight, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Early lysosomal maturation deficits in microglia triggers enhanced lysosomal activity in other brain cells of progranulin knockout mice

doi: 10.1186/s13024-018-0281-5

Figure Lengend Snippet: Cathepsins are elevated in the microglia depleted cell fraction of aged Grn −/− mice. a-d Cathepsin expression and maturation in the CD11b-positive, microglia enriched, fraction and the CD11b-negative, microglia depleted cellular fraction isolated form cortices of brains from 12-month-old Grn +/+ (wt) and Grn −/− (ko) mice. Representative immunoblots for the expression of CatD ( a, b ), CatB ( c, d ). The molecular weight standards in kilo Daltons (kDa) are indicated on the left side of all immunoblots. A dotted line in the blot indicates that samples of heterozygous mice were cut out, but all samples were loaded on one gel. Quantification of immunoblots for total cathepsin or pro-form (p), single chain (sc) and heavy chain (hc) normalized to wt are shown as mean ± SD. For statistical analysis the unpaired, two-tailed student’s t-test was used to compare ko to wt mice ( n = 3–5) (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant) ( a-d )

Article Snippet: CD11b-positive microglia were magnetically labelled with CD11b MicroBeads, loaded onto a MACS Column (Miltenyi Biotec) and subjected to magnetic separation.

Techniques: Expressing, Isolation, Western Blot, Molecular Weight, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Early lysosomal maturation deficits in microglia triggers enhanced lysosomal activity in other brain cells of progranulin knockout mice

doi: 10.1186/s13024-018-0281-5

Figure Lengend Snippet: Enhanced accumulation of lysosomal proteins in microglia of 3-month-old Grn −/− mice. Representative blots of LAMP1 ( a, b ) and saposin D (SapD) ( c, d ) in the CD11b-positive, microglia enriched fraction and the CD11b-negative, microglia depleted fraction isolated form brain cortices of 3-month-old Grn +/+ (wt) and Grn −/− (ko) mice. A dotted line in the blot indicates that samples of heterozygous mice were cut out, but all samples were loaded on one gel. Data were normalized to the corresponding mean value of wt mice and are shown as mean ± SD. For statistical analysis the unpaired, two-tailed student’s t-test was used to compare ko to wt mice ( n = 4–5) (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant)

Article Snippet: CD11b-positive microglia were magnetically labelled with CD11b MicroBeads, loaded onto a MACS Column (Miltenyi Biotec) and subjected to magnetic separation.

Techniques: Isolation, Two Tailed Test