Calcein

Cell pellet was resuspended in 200–3000 uL of Red Blood Cell Lysis Solution (ACK lysis buffer), depending on the pellet size. After incubation for 2 minutes at room temperature the ACK buffer was diluted 10-times with 1X PBS containing 2.5% FBS and pelleted again. Cell pellet was resuspended in 100 uL of 1X PBS + 2.5% FBS, mixed with 5 uL of Human TruStain FcX (Biolegend #422302), 3 uL of PE CD45 antibody (Biolegend # 368510 and 0.1 uL of calcein (1μg/μL, calcein (Biolegend #425201)), and left for 15 minutes on ice. Stained samples were washed twice with 2 ml of 1X PBS + 2.5% FBS, and finally resuspended in the same buffer supplemented with DAPI dye. Using BD FACSAria (BD Biosciences) or Sony MA900 (Sony) flow cytometers, cells were sorted on DAPI-, calcein+ (FITC+) to select for live cells. In addition, we sorted CD45+ (immune cells) and CD45− (cell population enriched in cancer cells) populations into separate tubes, and mixed them back in an artificial ratio to balance the compartmental representation (1:5–1:10 ratio, depending on cell availability). To define the percentage of immune cells in each sample, we registered the fraction of CD45+ and CD452210032 in the live cell (DAPI-, calcein+) population.

Triple negative breast cancer immune cells were processed as previously described [12 (link)]. Briefly, fresh TNBC core biopsy tumor specimens were received in MACS Tissue Storage Solution (Cat #130-100-008) on ice and minced in 10 cm ultra-low attachment dishes (Cat # 3262, Corning), and digested with Rat tail collagenase IV (Cat # 17104019, Life Technologies) for 30 min. Red blood cells were lysed with ACK buffer (Cat # 420301, BioLegend) and single cells were obtained and stained with DAPI (Cat # 422801, Biolegend) and Calcein (Cat # 425201, Biolegend). The BD Aria III sorter was used with a 100 μm nozzle to sort live (DAPI- Calcein+) single cells.