Journal: STAR Protocols

Article Title: Protocol for isolating stromal cells from lymphoid tissue for performing scRNA-seq

doi: 10.1016/j.xpro.2026.104501

Figure Lengend Snippet: Composition of cells recovered after each digestion step Collected cells were analyzed after each digestion step to determine cell phenotypes, numbers and viability. When performing transcriptomics analysis all digestion fractions will be combined. (A) Percentages of immune cells (CD45 + cells) and stromal cells (CD45 - cells) extracted from each digestion step analyzed via flow cytometry. (B) Representative viability images of each digestion step acquired on automated cell counter (LUNA 7-FX™) where red indicates dead cells and green live cells. (C) Immunofluorescence images of cells stained after each digestion step for CD45 (yellow) and Nuclei (blue). Representative Cytospin slides. Red arrows showing examples of CD45 - cells. Scale bars indicate 100 μm for main images and 10 μm for zoom. (D) After each digestion step the (i) percentage of live cells, (ii) number of live cells after each digestion measured by automated cell counting and (iii) percentage of immune (gray) and stromal cells (red) after each digestion fraction. (E) Combined cells from all digestions showing percentage and number of cells from each lymph node. (F) Representative gating strategy for lymph node cells showing stromal cell percentages. (G–L) Number of (G) immune cells, (H) stromal cells, (I) fibroblastic reticular cells (FRC), (J) lymphatic endothelial cells (LEC), (K) blood endothelial cells (BEC) and (L) double negative cells (DNC).

Article Snippet: CD45 antibody, anti-mouse, Biotin (Dilutions in 1:50) , Miltenyi Biotec , Cat# 130-124-209, RRID: AB_2819580.

Techniques: Transcriptomics, Flow Cytometry, Immunofluorescence, Staining, Cell Counting

Journal: STAR Protocols

Article Title: Protocol for isolating stromal cells from lymphoid tissue for performing scRNA-seq

doi: 10.1016/j.xpro.2026.104501

Figure Lengend Snippet: Cell selection using automated magnetic cell sorting (A) Cells were stained with CD45-biotin and CD31-biotin and sorted using autoMACS® Pro Separator. (B) Number of cells before staining for autoMACS® separation (step 11), and number of cells recovered from positive selection (CD45 + and CD31 + cells) and negative selection (CD45 - and CD31 - cells) in step 23. Each dot represents combined numbers from inguinal, axillary and brachial lymph nodes from 6 mice. Colors indicate biological replicates. (C) Percentages of cells after separation compared to the pre-staining cell count performed in step 11. (D) Purity check of separated cells using flow cytometry and staining for CD45 and CD31. (E) Overlay plots of positive selection (blue) and negative selection (red). (F) Percentage of CD45 + , CD45 - and CD31 + cells recovered (∗∗∗∗ p value < 0.0001, ∗ p value < 0.05). (G) Final viability check of positive and negative selected cells acquired just before performing scRNA-sequencing analysis.

Article Snippet: CD45 antibody, anti-mouse, Biotin (Dilutions in 1:50) , Miltenyi Biotec , Cat# 130-124-209, RRID: AB_2819580.

Techniques: Selection, FACS, Staining, Cell Characterization, Flow Cytometry, Sequencing

Journal: iScience

Article Title: A distinct TCR + CD19 + population in the peritoneal cavity: B1a cells as precursors of extrathymic T cells

doi: 10.1016/j.isci.2026.115505

Figure Lengend Snippet: Adoptively transferred B1a cells differentiate into DP cells and extrathymic T cell lineages (A) Two weeks after the adoptive transfer of B1a cells into muMT mice, the frequencies of TCR + CD19 + DP cells and B1a cells were assessed in the PEC, spleen, liver, small intestine, and thymus by flow cytometry. (B) Four weeks after the adoptive transfer of CD45.2 + B1a cells into CD45.1 + recipients, flow cytometric analysis was performed to evaluate the frequencies of CD45.2 + extrathymic T cells, DP cells, and B1a cells in the indicated tissues.

Article Snippet: CD45.1 + CD3 + T cells were isolated from mouse spleens, and CD45.2 + CD19 + B cells were isolated from the peritoneal cavity using magnetic-activated cell sorting (MACS) with anti-biotin microbeads (130-097-046; Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Adoptive Transfer Assay, Flow Cytometry

Journal: iScience

Article Title: Immune hub genes and a proof-of-concept prognostic signature in EBV-associated gastric carcinoma

doi: 10.1016/j.isci.2026.115243

Figure Lengend Snippet: Expression and immune correlation of hub genes (A) Violin plots show the expression levels of PTPRC and ITGB2 in EBVaGC (blue) and EBVnGC (red) from the TCGA-STAD cohort. Statistical significance is indicated (∗ p < 0.05 and ∗∗ p < 0.01). (B) Correlation heatmap illustrates the relationships between hub genes (PTPRC and ITGB2) and 22 tumor-infiltrating immune cell subsets. Circle size and color intensity represent the strength of correlation, with orange indicating positive correlations and teal indicating negative correlations. (C) Scatterplots demonstrates correlations between PTPRC expression and infiltration levels of five immune cell types: B cells memory, CD4 + memory activated T cells, CD8 + T cells, M1 macrophages, and M2 macrophages. Pearson’s correlation coefficients (R) and p values are shown for each comparison. (D) Scatterplots show correlations between ITGB2 expression and infiltration levels of the same five immune cell types as in (C). Pearson’s correlation coefficients (R) and p values are indicated.

Article Snippet: Anti-CD45 (PTPRC), mouse monoclonal, clone PD7/26+2B11 , Zhongshan Golden Bridge Biotechnology (ZSGB-BIO) , Cat# Kit-0024.

Techniques: Expressing, Comparison

Journal: iScience

Article Title: Immune hub genes and a proof-of-concept prognostic signature in EBV-associated gastric carcinoma

doi: 10.1016/j.isci.2026.115243

Figure Lengend Snippet: Increased CD18+, CD45+, and CD68 + immune-cell infiltration in EBVaGC (A) Representative immunohistochemistry (IHC) micrographs for CD18, CD45, and CD68 in EBVaGC (top) and EBVnGC (bottom). Brown DAB indicates positive staining; hematoxylin counterstain. Images were acquired at 40× objective; scale bars, 50 μm. (B) Dot-and-box plots showing quantitative cell densities (cells/mm 2 ) of CD18 + , CD45 + , and CD68 + cells in EBVaGC versus EBVnGC. Each dot represents one case (EBVaGC n = 20; EBVnGC n = 20; per case, the mean of ≥3 non-necrotic high-power fields was used). Boxes show IQR, center line the median, whiskers the range. p values are from two-sided exact Wilcoxon rank-sum tests (CD18: ∗∗∗ p < 0.0001; CD45: ∗∗∗ p < 0.0001; and CD68: ∗∗∗ p < 0.0001). Units and statistics are indicated on the plots.

Article Snippet: Anti-CD45 (PTPRC), mouse monoclonal, clone PD7/26+2B11 , Zhongshan Golden Bridge Biotechnology (ZSGB-BIO) , Cat# Kit-0024.

Techniques: Immunohistochemistry, Staining

Journal: iScience

Article Title: Immune hub genes and a proof-of-concept prognostic signature in EBV-associated gastric carcinoma

doi: 10.1016/j.isci.2026.115243

Figure Lengend Snippet: Construction and characterization of the four-gene immune score model in EBVaGC (A) Co-expression analysis of PTPRC and ITGB2 in EBVaGC samples. High-high expression pairs are highlighted in red. (B) GO biological process enrichment of immune genes co-expressed with PTPRC and ITGB2, highlighting terms such as leukocyte-mediated immunity and T cell activation. (C) KEGG pathway enrichment showing cytokine-cytokine receptor interaction, chemokine signaling, and other immune-related pathways. (D) LASSO Cox regression coefficients (lambda.min) for the selected prognostic genes GMPR and TIMD4. (E) Heatmap of GMPR and TIMD4 expression across risk groups. (F) Forest plot of multivariate Cox regression showing hazard ratios (HRs) and 95% confidence intervals for the four model genes. (G) GO enrichment of the final model genes, revealing involvement in phagocytosis, glial cell activation, and inflammatory response. (H) KEGG enrichment of model genes, with pathways such as cell adhesion molecules and primary immunodeficiency.

Article Snippet: Anti-CD45 (PTPRC), mouse monoclonal, clone PD7/26+2B11 , Zhongshan Golden Bridge Biotechnology (ZSGB-BIO) , Cat# Kit-0024.

Techniques: Expressing, Activation Assay