Fig 2: Composition of cells recovered after each digestion stepCollected 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).

Fig 3: 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.