apc cy7 cd45 1  (Thermo Fisher)

Journal: Cell Stem Cell

Article Title: Controlling genetic heterogeneity in gene-edited hematopoietic stem cells by single-cell expansion

doi: 10.1016/j.stem.2023.06.002

Figure Lengend Snippet: Single-cell cloning of gene-edited functional HSCs (A) Schematic showing the extracellular domain of CD45 with allele-specific antibody clones 104 and A20 and the epitope-defining amino acid. (B) Experimental setup of the single-cell editing and expansion experiment. (C) Left: fractions of CD201 + CD150 + KSL cells in single-cell-derived cultures 14 days after cloning (n = 261 clones). Right: Histogram of CD201 + CD150 + KSL cell frequency. Zoomed-in region shows clones with >10% CD201 + CD150 + KSL cells. (D and E) CD45.1 + donor PB chimerism (D) and lineage distribution (E ) in single recipients with long-term (LT) engraftment ≥5% and multilineage reconstitution (n = 8). Numbers over graphs in (D) represent percentage of CD201 + CD150 + KSL cells in the transplanted clone (%). (F) Linear correlation plots of CD201 + CD150 + KSL cell frequency and 16-week donor chimerism. Red dots indicate LT repopulating and multilineage clones. Pearson correlation. (G) CD45.1 + PB chimerism and lineage distribution in secondary recipients (n = 5). See also Figure S4 and Table S3 . Error bars represent SD.

Article Snippet: anti-mouse CD45.1-APC/Cy7 (A20) , Tonbo Biosciences , Cat#25-0453; RRID: AB_2621629.

Techniques: Cloning, Functional Assay, Clone Assay, Derivative Assay

Journal: Cell Stem Cell

Article Title: Controlling genetic heterogeneity in gene-edited hematopoietic stem cells by single-cell expansion

doi: 10.1016/j.stem.2023.06.002

Figure Lengend Snippet:

Article Snippet: anti-mouse CD45.1-APC/Cy7 (A20) , Tonbo Biosciences , Cat#25-0453; RRID: AB_2621629.

Techniques: Recombinant, Sequencing, Software, CRISPR

Journal: Frontiers in Medicine

Article Title: Conjunctival infiltrates and cytokines in an experimental immune-mediated blepharoconjunctivitis rat model

doi: 10.3389/fmed.2023.1200589

Figure Lengend Snippet: Conjunctival infiltrates in BC rats. (A) Total number of CD45 + 7AAD − cells per conjunctiva from 24 and 96h BC rats. (B–E) Percentage of conjunctival infiltrates subpopulations by flow cytometry. The Y-axis refers to the percentage of total CD45 + 7AAD − cells. Error bars: SD; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Control, PBS challenged group.

Article Snippet: APC-Cy7-CD45 (clone OX-1), BV421-CD3 (clone 1F4), and FITC-rat granulocyte-his48 were obtained from BD Pharmingen, Singapore.

Techniques: Flow Cytometry

Journal: Nature Immunology

Article Title: PI16 + reticular cells in human palatine tonsils govern T cell activity in distinct subepithelial niches

doi: 10.1038/s41590-023-01502-4

Figure Lengend Snippet: a , b , Representative flow cytometric plots depicting the gating strategy for each stromal cell type according to the indicated markers. c , Quantification of epithelial cells (EpC) as a percentage of total CD45 – CD235a – live cells in pediatric ( n = 5) and adult ( n = 6) patients with OSA. Data show average value of left and right tonsils for each patient. Mean and s.d. are indicated. P value was calculated with the two-sided Mann-Whitney test. d , Representative flow cytometric plots depicting the sorting strategy for scRNA-seq of CD45 – CD235a – stromal cells from human palatine tonsils according to the indicated markers. e – g , Expression patterns of indicated BEC, LEC and EpC gene signatures projected onto UMAPs. ScRNA-seq data represents a total of 86,966 CD45 – CD235a – stromal cells from n = 12 patients.

Article Snippet: Anti-human CD45–APC-Cy7 (clone 2D1, 560178, 1:100), anti-human CD4–BUV395 (clone SK3, 563550, 1:200), anti-human CD8–BUV805 (clone SK1, 612889, 1:400) and anti-human CD25–BUV563 (clone 2A3, 612918, 1:400) were purchased from BD Biosciences.

Techniques: MANN-WHITNEY, Expressing

Journal: Nature Immunology

Article Title: PI16 + reticular cells in human palatine tonsils govern T cell activity in distinct subepithelial niches

doi: 10.1038/s41590-023-01502-4

Figure Lengend Snippet: a , Representative UMAPs of distinct tonsillar stromal cell types according to the PhenoGraph clustering algorithm based on forward scatter (FSC)-A, side scatter (SSC)-A, CD31, ACTA2, PDPN and UEA1 flow cytometry data of CD45 − CD235a − cells ( n = 3 pediatric and n = 3 adult patients with OSA). Epithelial cells (EpCs), ACTA2 + cells, BECs, LECs, FRCs and negative cells (N) are indicated. b , UMAPs show the expression pattern of the indicated markers. c – e , Quantification of the indicated stromal cell types as a percentage of CD45 − CD235a − live cells in pediatric ( n = 5) and adult ( n = 6) patients with OSA. Data show average values of left and right tonsils for each patient. Mean and s.d. are indicated. P values were calculated with the two-sided Mann–Whitney test.

Article Snippet: Anti-human CD45–APC-Cy7 (clone 2D1, 560178, 1:100), anti-human CD4–BUV395 (clone SK3, 563550, 1:200), anti-human CD8–BUV805 (clone SK1, 612889, 1:400) and anti-human CD25–BUV563 (clone 2A3, 612918, 1:400) were purchased from BD Biosciences.

Techniques: Flow Cytometry, Expressing, MANN-WHITNEY

Journal: Nature Immunology

Article Title: PI16 + reticular cells in human palatine tonsils govern T cell activity in distinct subepithelial niches

doi: 10.1038/s41590-023-01502-4

Figure Lengend Snippet: a , Schematic representation of the scRNA-seq workflow. b , UMAP showing 14 CD45 − CD235a − stromal cell clusters after removal of contaminating cells including BECs, LECs, epithelial cells, ACTA2 + cells and FRCs. c , Dot plot depicts marker genes used for characterization and assignment of the indicated stromal cell types. Frames highlight marker genes of FRC clusters and the two ACTA2 + clusters (8 and 9). SMC, smooth muscle cell; SkMC, skeletal muscle cell. d , e , Expression patterns of VSMC and FRC gene signatures projected onto UMAPs. f , UpSet plot showing differentially expressed genes shared between VSMCs, PRCs in cluster 8 and FRCs. g , i , Top significantly enriched terms according to gene ontology (GO) enrichment analysis based on differentially expressed genes shared between the indicated cell types. Bar plots show counts of genes assigned to respective cellular processes. h , j , Expression patterns of genes assigned to the indicated cellular processes projected onto UMAPs. scRNA-seq data represent a total of 86,966 CD45 − CD235a − stromal cells from n = 12 patients. ECM, extracellular matrix.

Article Snippet: Anti-human CD45–APC-Cy7 (clone 2D1, 560178, 1:100), anti-human CD4–BUV395 (clone SK3, 563550, 1:200), anti-human CD8–BUV805 (clone SK1, 612889, 1:400) and anti-human CD25–BUV563 (clone 2A3, 612918, 1:400) were purchased from BD Biosciences.

Techniques: Marker, Expressing

Journal: Nature Immunology

Article Title: PI16 + reticular cells in human palatine tonsils govern T cell activity in distinct subepithelial niches

doi: 10.1038/s41590-023-01502-4

Figure Lengend Snippet: a – c , UMAPs display scRNA-seq data of tonsillar CD45 − CD235a − stromal cells separated and colored according to different conditions. FRCs showing strong transcriptional changes are highlighted by arrows. d , Bar plot shows the relative abundance of FRCs among CD45 − CD235a − stromal cells according to different conditions as revealed by scRNA-seq. w/o, without. e , UMAP visualization of re-embedded FRC subsets. FDC, follicular dendritic cells. f , Feature UMAPs show expression pattern of cluster marker genes used for characterization of the indicated FRC subsets. g , Chord diagram shows the proportion of cells derived from different conditions for each FRC subset. h , Diffusion map dimensionality reduction of FRC subsets. i , Top significantly enriched GO terms in PI16 + RCs according to enrichment analysis based on subset marker genes. Expression pattern of genes assigned to the indicated cellular processes is projected onto diffusion maps. BMP, bone morphogenic protein. scRNA-seq data represent a total of 86,966 CD45 − CD235a − stromal cells and 28,571 FRCs containing 20,158 cells (4,867 FRCs) from n = 3 pediatric patients with OSA, 21,596 cells (6,184 FRCs) from n = 5 adult patients with OSA and 45,212 cells (17,520 FRCs) from n = 4 adult patients with tonsillitis.

Article Snippet: Anti-human CD45–APC-Cy7 (clone 2D1, 560178, 1:100), anti-human CD4–BUV395 (clone SK3, 563550, 1:200), anti-human CD8–BUV805 (clone SK1, 612889, 1:400) and anti-human CD25–BUV563 (clone 2A3, 612918, 1:400) were purchased from BD Biosciences.

Techniques: Expressing, Marker, Derivative Assay, Diffusion-based Assay

Journal: Nature Immunology

Article Title: PI16 + reticular cells in human palatine tonsils govern T cell activity in distinct subepithelial niches

doi: 10.1038/s41590-023-01502-4

Figure Lengend Snippet: a , Bar plots show the relative abundance of indicated stromal cell types among CD45 – CD235a – cells of individual patients according to different conditions and based on scRNA-seq data. Mean and SEM are indicated. P values as per Kruskal-Wallis test. ScRNA-seq data represents a total of 86,966 CD45 – CD235a – stromal cells containing 20,158 cells from n = 3 pediatric patients with OSA, 21,596 cells from n = 5 adult patients with OSA and 45,212 cells from n = 4 adult patients with tonsillitis. b , UMAP showing 126,320 CD45 – CD235a – stromal cells from 12 patients acquired at KSSG (Kantonsspital St.Gallen, St.Gallen, Switzerland) and 3 patients independently acquired at UPENN (University of Pennsylvania, Philadelphia, USA) (see Extended Data Table ) integrated over their origin. BECs, LECs, EpCs, ACTA2 + cells and PI16 + RCs as a fraction of FRCs are indicated. c , UMAP visualization of tonsillar stromal cells split by sample origin. d , Bar plot showing the relative abundance of PI16 + RCs among CD45 – CD235a – cells per condition and based on scRNAseq data. Data represents n = 5 (Pediatric,OSA), n = 6 (Adult,OSA) and n = 4 (Adult,Tonsillitis) individual patients. Mean and SEM are indicated. P values as per two-sided Wilcoxon test. e , Heatmap showing the average expression of marker genes used for characterization of FRC subsets. f , Featureplots visualizing the expression pattern of indicated cytokines and chemokines across FRCs. g , UMAP depicting re-embedded FRCs colored according to the indicated patient groups. h , Significantly enriched terms according to GO enrichment analysis based on differentially expressed genes for the indicated FRC subsets. e-h , ScRNA-seq data represents 28,571 FRCs containing 4,867 cells from n = 3 pediatric patients with OSA, 6,184 cells from n = 5 adult patients with OSA and 17,520 cells from n = 4 adult patients with tonsillitis.

Article Snippet: Anti-human CD45–APC-Cy7 (clone 2D1, 560178, 1:100), anti-human CD4–BUV395 (clone SK3, 563550, 1:200), anti-human CD8–BUV805 (clone SK1, 612889, 1:400) and anti-human CD25–BUV563 (clone 2A3, 612918, 1:400) were purchased from BD Biosciences.

Techniques: Expressing, Marker

Journal: Frontiers in Immunology

Article Title: Dll1 Can Function as a Ligand of Notch1 and Notch2 in the Thymic Epithelium

doi: 10.3389/fimmu.2022.852427

Figure Lengend Snippet: T-cell development from Notch2 -deficient BM cells in the thymus of iD4 and iD1 mice. Age-matched control (WT) or Notch2 -deficient (N2KO) BM cells were obtained from Notch2 f/f or Rosa26 CreERT2 Notch2 f/f mice (CD45.1) 1 week after the administration of tamoxifen. BM chimeric mice were prepared in irradiated (6 Gy) C57 BL/6 mice (CD45.2) with Notch2 -deficient BM and analyzed 4 weeks after the reconstitution ( Supplementary Figure 3A ). (A) Flow cytometric analysis was performed using the thymocytes from BM chimeric mice with control (WT) or Notch2 -deficient (N2KO) BM cells ( Donor ) in Dll4 f/f (Cont), Dll4 f/f FoxN1-Cre (D4KO), iD4 Dll4 f/f FoxN1-Cre (iD4/D4KO), or iD1 Dll4 f/f FoxN1-Cre (iD1/D4KO) mice as the recipients ( Recipient ). Numbers in the profiles indicate the relative percentages, in CD45.1 + cells (left panels, CD19 vs. Thy1.2) and CD45.1 + Thy1.2 + cells (right panels, CD4 vs. CD8), for each quadrant or fractions. (B) Thymic cellularity (mean ± SD) of BM chimeric mice in Dll4 f/f (Cont, n=3), Dll4 f/f FoxN1-Cre (D4KO, n=3), iD4 Dll4 f/f FoxN1-Cre (iD4/D4KO, n=3), or iD1 Dll4 f/f FoxN1-Cre (iD1/D4KO, n=3) mice is shown. There are no statistically significant differences found between control and Notch2 -deficient BM cells by Student’s t -test. Each closed circle indicates the number of thymocytes (CD45.1) in each mouse. (C) Representative CD21/CD23 profiles in the donor-derived B cells (CD45.1 + B220 + ) obtained from the spleen of BM chimeric mice with control (WT) or Notch2 -deficient (N2KO) BM cells in Dll4 f/f mice are shown. The red polygons and numbers in the profiles indicate the MZB cell fraction and their frequencies. Results represent three independent biological replicates (A, C) .

Article Snippet: PE/Cy7-CD19 and APC/Cy7-CD45.1 (Thermo Fisher Scientific).

Techniques: Irradiation, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Dll1 Can Function as a Ligand of Notch1 and Notch2 in the Thymic Epithelium

doi: 10.3389/fimmu.2022.852427

Figure Lengend Snippet: T-cell development from Notch1 -deficient BM cells in the thymus of iD4 and iD1 mice. The primary BM chimeras were prepared in irradiated WT (CD45.2) mice with BM cells from Notch1 f/f (WT, CD45.1) or Rosa26 CreERT2 Notch1 f/f (N1KO, CD45.1) mice and GFP Tg mice. The control and Notch1 -deficient BM cells were obtained from the primary BM chimeras after the administration of tamoxifen and serially transferred into Dll4 f/f (Cont), Dll4 f/f FoxN1-Cre (D4KO), iD4 Dll4 f/f FoxN1-Cre (iD4/D4KO), or iD1 Dll4 f/f FoxN1-Cre (iD1/D4KO) mice. Four weeks after the reconstitution, thymocytes were analyzed ( Supplementary Figure 3B ). (A) Flow cytometric analysis was performed using the thymocytes from the secondary BM chimeric mice. Numbers in the profiles indicate the relative percentages, in CD45.1 + or GFP + cells (internal control, Int. cont) (left panels, CD19 vs. Thy1.2) and CD45.1 + Thy1.2 + or GFP + Thy1.2 + cells (right panels, CD4 vs. CD8), for each quadrant or fractions. Results represent at least three independent biological replicates. (B) The efficiencies of the appearance of CD4 + CD8 + (DP) thymocytes derived from control (WT) or Notch1 -deficient (N1KO) BM cells were examined. DP appearance index was calculated as the ratio of CD45.1 + /GFP + DP thymocytes and CD45.1 + /GFP + B220 + B cells in lymph node (mean ± SD; WT as donor; Cont, n=3; D4KO, n=3; iD4/D4KO, n=3; iD1/D4KO, n=4; N1KO as donor; Cont, n=5; D4KO, n=5; iD4/D4KO, n=5; iD1/D4KO, n=5). The data were collected from three independent experiments. Each closed circle indicates the index in each mouse. **p < 0.01, ***p < 0.001 by Mann–Whitney U -test.

Article Snippet: PE/Cy7-CD19 and APC/Cy7-CD45.1 (Thermo Fisher Scientific).

Techniques: Irradiation, Derivative Assay, MANN-WHITNEY

Journal: Journal of Inflammation Research

Article Title: Regulation of the Immune Microenvironment by an NLRP3 Inhibitor Contributes to Attenuation of Acute Right Ventricular Failure in Rats with Pulmonary Arterial Hypertension

doi: 10.2147/JIR.S336964

Figure Lengend Snippet: Flow Cytometry Antibody

Article Snippet: CD45 , 561586 , Rat CD45 APC-Cy7 OX-1 50ug , BD Pharmingen.

Techniques: Flow Cytometry, Marker

Journal: Journal of Inflammation Research

Article Title: Regulation of the Immune Microenvironment by an NLRP3 Inhibitor Contributes to Attenuation of Acute Right Ventricular Failure in Rats with Pulmonary Arterial Hypertension

doi: 10.2147/JIR.S336964

Figure Lengend Snippet: Changes in the proportion of immune cells in myocardial tissue were analysed via flow cytometry. ( A ) The proportion of CD45 + cells in each group was detected by flow cytometry. ( B ) The proportion of CD45 + CD11b + cells in each group was detected by flow cytometry. ( C ) The proportion of CD68 + CD86 + cells in each group was detected by flow cytometry. ( D ) Pulmonary hypertension and LPS caused a significant increase in the proportion of CD45-positive inflammatory cells in right ventricular tissue (normal vs PAH, normal vs normal + LPS, n = 3). ( E ) CD45-positive/CD11b-positive mononuclear macrophage infiltration was significantly elevated in right ventricular tissues from rats subjected to PAH or LPS (normal vs PAH, normal vs normal + LPS, normal + LPS vs PAH + LPS, n = 3). ( F ) The proportion of CD68-positive/CD86-positive M1 macrophages was decreased in the hearts of PAH rats and significantly increased in the right ventricular tissues of PAH rats stimulated with LPS, but MCC950 application significantly inhibited the change in the proportion of CD68-positive/CD86-positive M1 macrophages (n = 3). The significance of the difference was analysed by one-way ANOVA, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: CD45 , 561586 , Rat CD45 APC-Cy7 OX-1 50ug , BD Pharmingen.

Techniques: Flow Cytometry