Cyan adp 9 color

For membrane labeling, PBMC and TILs were stained with fluorochrome-coupled mAbs (detailed in Supplementary Table 8), incubated for 20 min at 4°C and washed. Cell samples were acquired on a Cyan ADP 9-color (Beckman Coulter), BD FACS Canto II flow-cytometers or on an 18-color BD LSRII (BD Biosciences) with single-stained antibody-capturing beads used for compensation (Compbeads, BD Biosciences or UltraComp eBeads, eBiosciences). Data were analyzed with FlowJo software v7.6.5 or v10 (Tree Star, Ashland, OR, USA).

All flow cytometry analyses were performed at the Colorado State University Animal Cancer Center on a Beckman Coulter CyAN ADP 9 Color analyzer running Summit Version 3.0 flow cytometry analysis software. Mammary epithelial stem cells were identified based on expression of CD44⁺/CD24^−/low immunotype and stem-like properties were confirmed utilizing the ALDEfluro Assay (Stem Cell Technologies). Monolayer MCF-7 and MCF-10A mammary epithelial cell cultures were stained for CD44 and CD24 expression. Briefly, ~3 × 10⁵ cells were dissociated from cell culture surface using 0.25% Trypsin-EDTA, pelleted, washed, and re-suspended in 30 μL of Flow Cytometry wash buffer (1X PBS, 1% FBS, and 1% Penicillin/Streptomyocin). Six microliters of direct FITC-conjugated mouse monoclonal anti-human CD44 antibody (BD Pharmingen #555478) and 6 μL of direct PE-conjugated mouse monoclonal anti-human CD24 antibody (BD Pharmingen #555428). Cells were then incubated for 30–60 min in the dark at 4°C. Following incubation, cells were pelleted and re-suspended in 500 μl of cold 1 × PBS and kept on ice until analysis. Analysis gates were established using cells from an unstained control and anti-mouse Ig,κ antibody capture beads (BD Pharmingen #552843).

For flow cytometry, whole-blood specimens were processed within 4 hours of collection to isolate PBMCs, using Lymphoprep (Axis-Shields-Diagnostics) as previously described [27 (link)]. Cells were analyzed using a CyAn ADP 9 color flow cytometer (Beckman Coulter). The T-cell panel included CD3 BV510, CD4 V450, CD38 PE Cy7, HLA-DR AF700, PD-1 APC, and CD57 FITC (all from BD Biosciences) and CD8 PE (Biolegend). The monocyte panel included HLA-DR AF700, CD14 PE Cy7, and CD16 PE (all from BD Biosciences). Anti-mouse Igk isotype control and negative control particles (BD Biosciences) were used for compensation. A standardized gating strategy was followed for T cells (Supplementary Figure 1A) and monocytes (Supplementary Figure 1B). Monocyte subsets were identified as previously described [28 (link)].