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: bioRxiv

Article Title: Local IFNγ signaling contributes to the regenerative decline of aged alveolar progenitor cells

doi: 10.64898/2026.04.07.716929

Figure Lengend Snippet: A) UMAP representation of subclustered lymphocytes in aging mouse lung (18 months) and young controls (3 months) scRNA-sequencing without injury. Whole murine lung aging dataset originally made public in Gote-Schniering et al. . B) UMAP labeled by old or young lung samples and (C) T cell abundance fold change in aged mouse lung by annotated cell type using milo analysis, excluding cell types that did not meet the threshold for quantification. D) IFNγ transcript via dotplot in lung lymphocytes separated by age, grouped into plots T cell/NK cell or B cell populations, and broken down by annotated cell type. E) Human lung cell atlas comparison of IFNγ response genes by GSEA of hallmark pathways in human CD8+ T cells and NK cells across age cohorts; young (<30 years), old (>60 years). F) A comparative dotplot of CD8+ T cell IFNγ gene expression in the same population of human lung cell atlas samples. G) Experimental diagram of retroorbital injection of anti-CD45 into old and young mice followed by lung flow cytometry to measure lung-resident lymphocyte populations for intracellular IFNγ. H) Abundance of key resident lung lymphocyte subpopulations and (I) resident IFNγ+ subpopulations. J) Gross pathology and immune cell infiltration shown by H&E stain in comparable old and young perivascular regions. K) Representative fluorescent imaging of bronchiolar/vascular regions in old and young lung using a combined immunofluorescence and RNAscope approach, including staining with anti-SPC for AT2 cells (green), IFNγ mRNA transcript (red), and CD3e mRNA transcript (white). Zoomed-in cutouts highlight IFNγ+ CD3e+ T cells within TLS in the distal lung, with examples marked by yellow arrows. “Aw” labels airways and “Endo” marks endothelial vessels. L) Quantification of total IFNγ+ CD3e+ T cells and CD3e+ T cells per mm 2 in perivascular/peribronchiolar lung regions. M) Quantitation of the density of puncta marking IFNγ mRNA transcript in CD3e+ cells compared by age and localization within TLS-like structures. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Stomal cells were isolated from 2–4-week-old neonatal mouse lungs (digested with dispase/collagenase as above), including negative selection with Anti-CD31 (Miltenyi Biotech #130-097-418), Anti-CD45 (Miltenyi Biotech #130-052-301), and Anti-EpCAM (Miltenyi Biotech #130-105-958) magnetic beads.

Techniques: Sequencing, Labeling, Comparison, Gene Expression, Injection, Flow Cytometry, Staining, Imaging, Immunofluorescence, RNAscope, Quantitation Assay