Pe cy7 annexin 5

Dissociated cancer cells were filtered through a 4 µm strainer and suspended in phosphate-buffered saline (PBS) supplemented with 2% FBS and 2 mM EDTA (fluorescence-activated cell sorting (FACS) buffer) as previously described^{11 (link)}. In all, 1 µL of mouse IgG (1 mg/mL) was added and incubated at 4 °C for 10 min. The cells were then re-suspended in 1× binding buffer and anti-CD44 (allophycocyanin) in combination with anti-CD24 (phycoerythrin (PE)) (BD, Mississauga, ON, Canada) antibodies were added according to the manufacturer’s instructions. The cells were washed twice with FACS buffer and 7-aminoactinomycin D (7-AAD, eBioscience, San Diego, CA) and Annexin-V/PE-Cy7 (eBioscience) was added and incubated for 15 min at room temperature to assess dead and apoptotic cells. Flow cytometry was performed on a Cyan-ADP 9 and the BD LSRFortessa. Data was analyzed with the FlowJo software (Ashland, OR, USA).

Cell suspensions of mouse tissues were prepared in RPMI-1640 (Wisent) with 2% (v/v) FCS, 100 μg/mL streptomycin and 100 U/mL penicillin (Wisent). The cells were stained for surface markers in PBS with 2% FCS for 20 min on ice, using antibodies listed in Supplemental Tables S5 and S6. Viability Dye eFluor® 506 (eBioscience) was used to discriminate dead cells. Annexin V PeCy7 (eBioscience) was used for detection of apoptotic cells. Compensation was performed with BD™ CompBeads (BD Biosciences). The data were acquired on BD Fortessa and analyzed with the FACS Diva (BD Biosciences) or the FlowJo (Tree Star) software.