Brdu apc

Flow cytometry analysis for proliferation or apoptosis was performed on TRAMP C2RE3 cells cultured in complete media or cultured in serum free media for 48 hours. For proliferation, cells were labeled with 10μM BrdU for 4 hours, stained with BrdU-APC (17-5071-41, eBiosciences) following the manufacturer’s instructions. For apoptosis, cells were stained with Annexin V-APC (550474, BD Biosciences) and 50 ng Propidium Iodide (556463, BD Biosciences) per manufacturer’s instructions. Flow cytometry was performed and analyzed using the LSRFortessa and FACS Diva software (BD Biosciences).

For co-culture apoptotic studies, R7 cells were labeled with Vybrant^™ DiO cell-labeling dye (Invitrogen). DiO-labeled R7 cells were co-cultured either under SFM conditions with TK^+/+ M2 or TK⁻/⁻ M2 BMDMs at a 1:4 ratio or in media containing a 1:1 ratio of M2 BMDM CM to SFM for 24 hours. Cells were stained with Annexin V-APC and Propidium Iodide and analyzed as described (3 (link), 8 (link), 27 (link)). Samples were normalized to M2 TK^+/+ BMDMs within respective treatment conditions. For proliferation, DiO-labeled R7 cells were co-cultured for 24 hours at a 1:4 ratio with TK^+/+ M2 BMDMs or TK^−/− M2 BMDMs or in media containing a 1:1 ratio of M2 BMDM CM to SFM, labeled with BrdU, and stained with BrdU-APC (eBiosciences). For both co-cultured proliferation and apoptosis assays, cells were gated on DiO-positivity to select out macrophages within the co-culture. All samples were normalized to TK^+/+ within respective treatment conditions (co-culture versus conditioned media). For BCa stem cell marker analyses, dissociated mammary tumor cells were stained with CD24-PE, CD29-FITC, CD31-APC, TER-119-APC (BD Biosciences) and CD45-APC (Biolegend) antibodies, analyzed, and sorted as previously described (8 (link)).