Fixative buffer

Blood cells collected via cardiac puncture were lysed of RBC via incubation with NH₄Cl buffer for 15 min at room temperature with gentle tilting. RBC lysis was inactivated with equal volume of 1xPBS followed by centrifugation at 450 g for 5 min at 4°C and resuspended in 1xPBS. Mammary tissues were digested as previously described until after the RBC lysis step in which mammary cells were subsequently reconstituted in 1xPBS. Staining of cells was carried out with a cocktail of primary antibodies from either BioLegend or eBioscience comprising of APC-Cy7-anti-CD45 (clone 30-F11), FITC-anti-Ly6C (clone HK1.4), PE-anti-Ly6G (clone 1A8), Biotin-anti-Gr1 (clone RB6-8C5), and BV605-anti-CD11b (clone M1/70). Secondary antibody staining was performed with Alexa Fluor 647-streptavidin antibody (BioLegend). Dead cells were stained with eFluor 450-fixable viability dye (eBioscience) before fixation with fixative buffer (BioLegend).

Staining of cells was carried out with a cocktail of primary antibodies on ice at 1:200 dilution from either BioLegend or eBioscience comprising of APC-Cy7-anti-CD45 (clone 30-F11), FITC-anti-Ly6C (clone HK1.4), PE-anti-Ly6G (clone 1A8), BV605-anti-CD11b (clone M1/70), FITC-anti-TCRγ/δ (clone GL3), PerCP/Cy5.5-anti-CD4 (clone GK1.5), and BV605-anti-CD8a (clone 53–6.7). Dead cells were stained with eFluor 450-fixable viability dye (eBioscience) at 1:1000 dilution before fixation with fixative buffer (BioLegend). The gating strategy of flow cytometry analysis is outline in Supplementary Figure 1A.

Fast-activated cell-based (FACE) assays were performed as we described previously [92 (link)]. SFs were seeded at 10⁴ cells/well (96-well plates) and after overnight incubation in 1% FCS DMEM to synchronize the cells, were then stimulated for the indicated time points (from 0 to 120 minutes) before being treated with fixative buffer (Biolegend) according to the manufacturer’s recommendations. Once permeabilized, the cells were incubated with antibodies specific for either the activated or whole protein form of the signalling element (e.g., ERK1/2 and the dually phosphorylated active forms of ERK [pERK; Cell Signalling Technology]) for 1 hour before addition of streptavidin-conjugated horseradish peroxidase for 30 minutes (anti-rabbit HRP, Cell Signalling Technology). Following washing and incubation with tetramethylbenzidine (TMB), absorbance was read at 450nm using a Tecan Sunrise plate reader.