easysep immunomagnetic cd19 positive selection kit  (STEMCELL Technologies Inc)

Journal: bioRxiv

Article Title: IFI16/AIM2 inflammasomes control Gal-9 and PVR in myeloid cells from PWH and their targeting improves immunotherapy against HIV-1

doi: 10.64898/2026.01.26.701473

Figure Lengend Snippet: (A): ELISA analysis of plasma concentration of IL-1β, comparing PWH with normal CD4/CD8 T cell ratio (defined as ≥1) and low CD4/CD8 T cell ratio (defined as < 0.8). Statistical significance was calculated using two-tailed Wilcoxon test. (B): Spearman correlation between plasma concentrations of soluble CD14 (ssCD14) and IL-1β. P and R values are shown (C): Expression of Gal-9 (Gal9; higher plot) or PVRhi (lower plot) in activated CD40hi in Mo from HIV- 1-negative controls, and PWH with low CD4/CD8 T cell ratio or normal CD4/CD8 T cell ratio after 16-hour stimulation with PAMPs mimicking bacterial translocation (LPS, red; Flagellin, Flag, purple) or viral replication (CL097, ssRNA, orange; Poly I:C, dsRNA, blue). Statistical significance was calculated using Kruskal-Wallis test for comparison of stimulation with the same PAMP between groups (*p<0.05; **p<0.01), and One-Way ANOVA Friedman test with Dunn’s multiple comparison test for comparison within the same group (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Article Snippet: Mo were isolated from PBMC from our study cohort by the human CD14 MicroBeads kit positive immunomagnetic selection using MS Columns (Miltenyi Biotec).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Concentration Assay, Two Tailed Test, Expressing, Translocation Assay, Comparison

Journal: bioRxiv

Article Title: IFI16/AIM2 inflammasomes control Gal-9 and PVR in myeloid cells from PWH and their targeting improves immunotherapy against HIV-1

doi: 10.64898/2026.01.26.701473

Figure Lengend Snippet: (A-B): Histological analysis of IFI-16 (A) or AIM2 (B) (green) and Caspase-1 (red) expression and DAPI (blue) in the spleen from a viremic HIV-1 infected humanized BLT mouse. Quantifications of cells positive cells for each inflammasome sensor alone (top plots), co-expressing Caspase 1 (CASP1) (middle plots) or both Caspase 1 and CD14 (bottom plots) are shown on the right. (C): Histological analysis of Gal-9 (Gal9; green), CD14 (white) and Caspase-1 (Casp1, red) expression and DAPI (blue) in the spleen from a viremic HIV-1 infected humanized BLT mouse. Zoomed area demonstrating co-expression between Gal9, CD14 and Caspase-1 are shown on the right. Arrows highlight cells co-expressing the mentioned markers

Article Snippet: Mo were isolated from PBMC from our study cohort by the human CD14 MicroBeads kit positive immunomagnetic selection using MS Columns (Miltenyi Biotec).

Techniques: Expressing, Infection

Journal: bioRxiv

Article Title: Tumor-infiltrating CD27 - IgD - regulatory B cells suppress cytotoxic CD8 + T cell responses in renal cell carcinoma

doi: 10.1101/2025.07.08.663720

Figure Lengend Snippet: (A) Representative contour plots and cumulative data showing the frequencies of CD19 + CD27 - IgD - (double negative (DN)), CD19 + CD27 + IgD - , CD19 + CD27 + IgD + (unswitched memory (USM)) and CD19 + CD27 - IgD + (naive) B cells ex vivo in paired BK and matching tumor tissue; ** p <0.01, *** p <0.001. n= 32 biologically independent samples. DN, USM, and naive B cells analyzed by paired two-tailed Student’s t -test, CD19 + CD27 + IgD - B cells analyzed by paired two-tailed Wilcoxon test. Error bars represented as mean±SEM. (B) Representative contour plots and cumulative data showing the frequencies of CD19 + CD24 lo/- CD38 hi (plasma cells (PCs)) and BLIMP-1 + B cells ex vivo in paired BK and matching tumor tissue. BLIMP-1 expression overlaid onto the B cell contour plot; ** p <0.01, **** p <0.0001. For plasma cells n =31 and for BLIMP-1 + B cells n =15 biologically independent samples. Paired two-tailed Wilcoxon test. Error bars represented as mean±SEM. (C) Representative contour plots and cumulative data showing the frequencies of CD19 + CD27 + IgD - CD24 + CD38 - IgM + (IgM + memory) and CD19 + CD27 + IgD - CD24 + CD38 - IgM - (class-switched memory (CSM)) B cells ex vivo in paired BK and tumor; *** p <0.001. n= 32 biologically independent samples. CSM analyzed by paired two-tailed Student’s t -test, IgM + memory analyzed by paired two-tailed Wilcoxon test. Error bars represented as mean±SEM. (D) Representative contour plots and cumulative data showing the frequencies of CD19 + CD27 - IgD + CD24 int CD38 int (mature) and CD19 + CD27 - IgD + CD24 hi CD38 hi transitional B cells ex vivo in paired BK and tumor. n= 32 biologically independent samples. Paired two-tailed Wilcoxon test. Error bars represented as mean±SEM. (E) Representative contour plots and cumulative data showing the frequencies of CD19 + CD27 - IgD - CD21 + CD11c - (DN1), CD19 + CD27 - IgD - CD21 - CD11c + (DN2), CD19 + CD27 - IgD - CD21 - CD11c - (DN3), and CD19 + CD27 - IgD - CD21 + CD11c + (DN4) B cells ex vivo in BK and tumor; ** p <0.01. n= 18 biologically independent samples. Paired two-tailed Student’s t -test. Error bars represented as mean±SEM. (F) Cumulative data showing the frequencies of CD19 + CD27 - IgD + (naive), CD19 + CD27 + IgD - , CD19 + CD27 - IgD - CD21 + CD11c - (DN1), CD19 + CD27 - IgD - CD21 - CD11c - (DN3) B cells, and CD19 + CD24 lo/- CD38 hi (plasma cells (PCs)) stratified according to intermediate (3-5) or high (>6) Leibovich scores; * p <0.05, ** p <0.01. For naive, CD27 + IgD - , and plasma cells n= 26, for DN1 and DN3 n= 13 biologically independent samples. Naive, DN1, DN3, and PC analyzed by unpaired two-tailed Student’s t -test, CD27 + IgD - analyzed by unpaired Mann-Whitney U test. Error bars represented as mean±SEM.

Article Snippet: Tissue-resident B cells were isolated using the EasySep immunomagnetic CD19 positive selection kit (STEMCELL, cat. no. 17854).

Techniques: Ex Vivo, Two Tailed Test, Clinical Proteomics, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Tumor-infiltrating CD27 - IgD - regulatory B cells suppress cytotoxic CD8 + T cell responses in renal cell carcinoma

doi: 10.1101/2025.07.08.663720

Figure Lengend Snippet: (A) Density UMAPs showing the distribution of IL10 hi , TGFB hi , TXN hi , AHR hi , HAVCR2 hi , TIGIT hi , CD274 hi and PDCD1 hi RCC-resident B cells. (B) Dotplot showing the proportion of cells expressing a Breg score comprising IL10, CD274, AHR, TXN, LAG3, TIGIT, CTLA4, CD86, TGFB1, and GZMB (dot size), and their average expression levels (color intensity) in the tumor, tumor margin, BK, and blood. (C) Spatial analysis of segmented CD20 + Bregs in a representative TLS. Staining of DAPI (dark blue) overlaid with cell segmentation (gating strategy shown in Figure S5A) of CD20 + IL-10 + Bregs (red), CD20 + TGFβ + Bregs (green), and CD20 + IL-10 + TGFβ + Bregs (orange). Scale bar = 100µm. (D) Cumulative data showing the frequencies of CD20 + IL-10 + Bregs, CD20 + TGFβ + Bregs, CD20 + IL-10 + TGFβ + within ROIs; * p <0.05, ** p <0.01. 13 ROIs across n =3 biologically independent samples. One-way ANOVA with Tukey’s test for multiple comparisons. Error bars represented as mean±SEM. (E) Spatial analysis of segmented CD138 + Bregs in a representative TLS. Staining of DAPI (dark blue) overlaid with cell segmentation (gating strategy shown in Figure S5A) of CD138 + IL-10 + Bregs (pink), CD138 + TGFβ + Bregs (dark green), and CD138 + IL-10 + TGFβ + Bregs (yellow) within TLSs. (F) Cumulative data showing the frequencies of CD138 + IL-10 + Bregs, CD20 + CD138 + TGFβ + Bregs, CD138 + IL-10 + TGFβ + Bregs within ROIs; * p <0.05, ** p <0.01. 13 ROIs across n =3 biologically independent samples. One-way ANOVA with Tukey’s test for multiple comparisons. Error bars represented as mean±SEM. (G) Cumulative data showing the frequencies of CD20 + CD27 - IgD + (naive), CD20 + CD27 + IgD + (unswitched memory (USM)), CD20 + CD27 + IgD - B cells, CD20 + CD27 - IgD - CD21 + CD11c - (DN1), CD20 + CD27 - IgD - CD21 - CD11c + (DN2), CD20 + CD27 - IgD - CD21 - CD11c - (DN3), and CD20 + CD27 - IgD - CD21 + CD11c + (DN4) within CD20 + IL-10 + Bregs; * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. 13 ROIs across n =3 biologically independent samples. Fridman test with Dunn’s test for multiple comparisons. Error bars represented as mean±SEM. (H) Cumulative data showing the frequencies of CD20 + CD27 - IgD + (naive), CD20 + CD27 + IgD + (unswitched memory (USM)), CD20 + CD27 - IgD - (double negative (DN)), CD20 + CD27 + IgD - B cells, CD20 + CD27 - IgD - CD21 + CD11c - (DN1), CD20 + CD27 - IgD - CD21 - CD11c + (DN2), CD20 + CD27 - IgD - CD21 - CD11c - (DN3), and CD20 + CD27 - IgD - CD21 + CD11c + (DN4) B cells within CD20 + TGFβ + Bregs; * p <0.05, ** p <0.01. 13 ROIs across n =3 biologically independent samples. Fridman test with Dunn’s test for multiple comparisons. Error bars represented as mean±SEM. (I) Prognostic association of high versus low CD19, IL-10, TGFB1 signature with survival in RCC using The Cancer Genome Atlas (TCGA) data. p values were derived by Cox proportional hazard model adjusting for signature group, age, sex, and American Joint Committee on Cancer (AJCC) stage.

Article Snippet: Tissue-resident B cells were isolated using the EasySep immunomagnetic CD19 positive selection kit (STEMCELL, cat. no. 17854).

Techniques: Expressing, Staining, Derivative Assay

Journal: bioRxiv

Article Title: Tumor-infiltrating CD27 - IgD - regulatory B cells suppress cytotoxic CD8 + T cell responses in renal cell carcinoma

doi: 10.1101/2025.07.08.663720

Figure Lengend Snippet: (A) Radar plot showing the Human Molecular Signatures Database and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched in each tumor-infiltrating B cell (TIB) cluster. (B) Density UMAPs showing the distribution of TLR7 hi , TLR9 hi , MYD88 hi , IRAK1 hi , IRAK4 hi , TRAF6 hi , MAP3K7 hi , and NFKB1 hi RCC-resident B cells. (C) Representative contour plots and cumulative data showing IL-10 expression by tumor-infiltrating CD19 + B cells ex vivo (white) and following 72h stimulation with R848 (light grey) or CpGC (dark grey); ** p <0.01, *** p <0.001. For ex vivo n =16, R848 and CpGC n =22 biologically independent samples. Mixed-effects one-way ANOVA with Dunnett’s test for multiple comparisons. Error bars represented as mean±SEM. (D) Representative contour plots and cumulative data showing TGFβ expression by tumor-infiltrating CD19 + B cells ex vivo (white) and following 72h stimulation with R848 (light grey) or CpGC (dark grey); * p <0.05, ** p <0.01. For ex vivo n =13, R848 and CpGC n =10 biologically independent samples. One-way Fridman test with Dunn’s test for multiple comparisons. Error bars represented as mean±SEM. (E) Representative contour plots and cumulative data showing the frequencies of CD19 + CD27 - IgD + (naive), CD19 + CD27 + IgD + (unswitched memory (USM)), CD19 + CD27 + IgD - , and CD19 + CD27 - IgD - (double negative (DN)) B cells within tumor-infiltrating CD19 + IL-10 + B cells, ex vivo and following 72h stimulation with R848 or CpGC; * p <0.05, ** p <0.01, **** p <0.0001. n =24 biologically independent samples. Two-way ANOVA with Tukey’s test for multiple comparisons. Error bars represented as mean±SEM. (F) Representative contour plots and cumulative data showing the frequencies of CD19 + CD27 - IgD - CD21 + CD11c - (DN1), CD19 + CD27 - IgD - CD21 - CD11c + (DN2), CD19 + CD27 - IgD - CD21 - CD11c - (DN3) and CD19 + CD27 - IgD - CD21 + CD11c + (DN4) B cells within tumor-infiltrating CD19 + IL-10 + B cells, ex vivo and following 72h stimulation with R848 or CpGC; * p <0.05, *** p <0.001, **** p <0.0001. n =18 biologically independent samples. Two-way ANOVA with Tukey’s test for multiple comparisons. Error bars represented as mean±SEM. (G) Representative contour plots and cumulative data showing the frequencies of CD19 + CD27 - IgD + (naive), CD19 + CD27 + IgD + (unswitched memory (USM)), CD19 + CD27 + IgD - , and CD19 + CD27 - IgD - (double negative (DN)) B cells within tumor-infiltrating CD19 + TGFβ + B cells, ex vivo and following 72h stimulation with R848 or CpGC; ** p <0.01, **** p <0.0001. n =12 biologically independent samples. Two-way ANOVA with Tukey’s test for multiple comparisons. Error bars represented as mean±SEM. (H) Representative contour plots and cumulative data showing the frequencies of CD19 + CD27 - IgD - CD21 + CD11c - (DN1), CD19 + CD27 - IgD - CD21 - CD11c + (DN2), CD19 + CD27 - IgD - CD21 - CD11c - (DN3) and CD19 + CD27 - IgD - CD21 + CD11c + (DN4) B cells within tumor-infiltrating CD19 + TGFβ + B cells, ex vivo and following 72h stimulation with R848 or CpGC; *** p <0.001, **** p <0.0001. n =11 biologically independent samples. Two-way ANOVA with Tukey’s test for multiple comparisons. Error bars represented as mean±SEM.

Article Snippet: Tissue-resident B cells were isolated using the EasySep immunomagnetic CD19 positive selection kit (STEMCELL, cat. no. 17854).

Techniques: Expressing, Ex Vivo

Journal: bioRxiv

Article Title: Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging

doi: 10.1101/2024.03.14.585083

Figure Lengend Snippet: A ) Schematic describing the sample collection and cell isolation strategy. Magnetic-activated cell sorting (MACS) separation of hematopoietic, stem/progenitor, and mesenchymal fractions were performed and then pooled into one scRNA-Seq reaction per patient. B ) UMAP representation of 82,742 single-cell transcriptomes from bone marrow of 12 individuals. AEC, arterial endothelial cell. SEC, sinusoidal endothelial cell. VSMC, vascular smooth muscle cell. Ba, Basophil. Eo, Eosinophil. Ma, Mast Cell. RBC, red blood cell. pDC, plasmacytoid dendritic cell. CLP, common lymphoid progenitor. MEP, megakaryocyte erythroid progenitor. GMP, granulocyte monocyte progenitor. MPP, multipotent progenitor. HSPC, hematopoietic stem and progenitor cell. HSC, hematopoietic stem cell. Meg/E, megakaryocyte/erythroid. MSC, mesenchymal stromal cell. C ) Bar plots showing the cell counts for each lineage captured (left) and the per sample cell lineage frequencies normalized by the total cells in each sample (right). D ) Heatmap with normalized gene expression scaled by column (cell type) showing the gene expression of key cell type marker genes. Rows were clustered such that genes expressed in similar cell types cluster together. The top two most significant genes by adjusted p-value comparing each cell type to all other cell types were plotted. Selected marker genes for each lineage were labeled. EC, endothelial cell. M, vascular smooth muscle.

Article Snippet: 20uL of RBC depleted cells were put on ice, while 67% of the remaining cells were used for immunomagnetic CD45 depletion using EasySep Human CD45 Depletion Kit II (STEMCELL # 17898) to isolate non-hematopoietic cells in an unbiased fashion, while 33% of cells were subjected to immunomagnetic CD34 enrichment using EasySep Human CD34 Positive Selection Kit II (STEMCELL #17856).

Techniques: Cell Isolation, FACS, Gene Expression, Marker, Labeling

Journal: bioRxiv

Article Title: Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging

doi: 10.1101/2024.03.14.585083

Figure Lengend Snippet: A ) UMAP computed from 19,257 mesenchymal cells from 12 individuals showing different mesenchymal subsets, with RNAlo MSCs being excluded. B ) Dot plot showing normalized expression of key mesenchymal marker genes in MSC subsets. Rows and columns were manually ordered. C ) Dot plot showing the normalized expression of literature-derived marker genes for human MSCs including NT5E (CD73), THY1 (CD90), and ENG (CD105). NGFR (CD271) and MCAM (CD146) have also been described as canonical MSC markers. D ) CytoTRACE analysis projected onto the MSC UMAP showing the predicted differentiation score, where higher values imply the cell is more primitive. E) Boxplots showing the relative colony forming potential (number of colonies produced by each cell type divided by total colonies from each sample, each data point is one sample) of sorted Fibro-MSC (CD45-CD38-CD235ab-VECAD-PDPN+), THY1+ MSC (CD45-CD38-CD235ab-VECAD-PDPN-LEPR+ CD90+), Adipo-MSC (CD45-CD38-CD235ab-VECAD-PDPN-LEPR+ CD90-), and osteolineage cells (CD45-CD38-CD235ab-VECAD-PDPN-CD56+). P-values were computed as Fibro-MSC vs. all by Dunnett’s Multiple Comparisons test. F) Line plot showing the population doublings over the course of cell culturing for eight passages, where all cells were passaged every 7 days. P-values are computed as Fibro-MSC vs other MSCs by two-way ANOVA. G ) UMAP showing 3,874 endothelial cells from the 12 individuals. SEC – Sinusoidal Endothelial Cells, AEC – Arterial Endothelial Cells. H ) Dot plot showing normalized expression of top differentially expressed genes by adjusted p-value between endothelial subsets, as well as CDH5 , CD34 , and KDR which were manually selected as pan-endothelial markers. I ) MSCs from human bone marrow aspirate reported from De Jong et al., Nature Immunology , 2021 were reference mapped to our scRNA-Seq atlas and the cell type labels were predicted. J ) Human fetal bone marrow mesenchymal cells from Jardine et al., Nature , 2021 were reference mapped to our scRNA-Seq atlas and the cell labels were predicted.

Article Snippet: 20uL of RBC depleted cells were put on ice, while 67% of the remaining cells were used for immunomagnetic CD45 depletion using EasySep Human CD45 Depletion Kit II (STEMCELL # 17898) to isolate non-hematopoietic cells in an unbiased fashion, while 33% of cells were subjected to immunomagnetic CD34 enrichment using EasySep Human CD34 Positive Selection Kit II (STEMCELL #17856).

Techniques: Expressing, Marker, Derivative Assay, Produced, Cell Culture

Journal: bioRxiv

Article Title: Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging

doi: 10.1101/2024.03.14.585083

Figure Lengend Snippet: A) Schematic depicting the CODEX experimental and computational workflow leading to cell type identification. B) Diagram showing the 54-plex CODEX panel (53 antibodies + DAPI) split by target cell population. C) Heatmap showing average centered-log-odds ratio normalized expression per cell type scaled and clustered by protein showing the marker protein expression is consistent with literature knowledge (Left). Cell types were annotated by unsupervised clustering and manual gating as described in Materials and Methods. Boxes were manually drawn to highlight coordinate marker expression. UMAP showing the 803,131 single cells in the CODEX atlas from 12 individuals colored by cell type (Right). Adipocytes were labeled with /Artifact because this population contained a mixture of true adipocytes and artifactual staining. HSPC – Hematopoietic Stem and Progenitor Cell. Meg/E-Megakaryocyte/Erythroid, Endo-Endothelial, M-Vascular Smooth Muscle, Sh-Schwann Cells. D) Stacked bar plot showing cell type frequencies normalized by total cells per sample. E) CODEX image (left) is paired with the cell phenotype map (CPM,right). An arteriolar structure and hematopoietic cells are shown using selected relevant fluorescent markers, which is juxtaposed with the same image with the cellular segmentation masks colored by cell type showing how CODEX allows single-cell mapping of the bone marrow microenvironment.

Article Snippet: 20uL of RBC depleted cells were put on ice, while 67% of the remaining cells were used for immunomagnetic CD45 depletion using EasySep Human CD45 Depletion Kit II (STEMCELL # 17898) to isolate non-hematopoietic cells in an unbiased fashion, while 33% of cells were subjected to immunomagnetic CD34 enrichment using EasySep Human CD34 Positive Selection Kit II (STEMCELL #17856).

Techniques: Expressing, Marker, Labeling, Staining