Pecy7 conjugated anti cd24

Two million to 5 million cells per condition were incubated in 250 µL of 2% FBS/PBS with fluorochrome-conjugated primary antibodies for 30 min with shaking every 10 min. Primary antibodies were washed with 2% FBS/PBS, and cells were resuspended in 2.5 mg/mL DAPI (Invitrogen) before analysis. The following primary antibodies were used for the analysis of K14rtTA/TetO-Cre/Rosa-YFP mice: PECy7-conjugated anti-CD24 (1:100; BD Biosciences, clone M1/69), APC-conjugated anti-CD29 (1:100; eBiosciences, clone eBioHMb1-1), PE-conjugated anti-CD45 (1:100; eBiosciences, clone 30-F11), PE-conjugated anti-CD31 (1:100; BD Biosciences, clone MEC 13.33), and PE-conjugated anti-CD140a (1:100; eBiosciences, clone APA5). Data analysis was performed on a BD LSR Fortessa using FACS DiVa software (BD Biosciences). Dead cells were excluded with DAPI; CD45⁺, CD31⁺, and CD140a⁺ cells were excluded (Lin⁺) before analysis of the YFP⁺ cells. Primary antibodies used for the analysis of K8rtTA/TetO-Cre/Rosa-tdTomato mice were PECy7-conjugated anti-CD24 (1:100; BD Biosciences, clone M1/69), FITC-conjugated anti-CD29 (1:100; BD Biosciences, clone Ha2/5), APC-conjugated anti-CD45 (1:100; eBiosciences, clone 30-F11), APC-conjugated anti-CD31 (1:100; eBiosciences, clone 390), and APC-conjugated anti-CD140a (1:100; eBiosciences, clone APA5). A minimum of five 10⁵ total events per mouse were analyzed.

For the analysis of CD24 and CD44 expression on cell membrane, 5 × 10⁵ of cells were collected in Cell Staining Buffer (BioLegend, #420201) and stained with PE-Cy™7-conjugated anti-CD24 (1:100 for 20 min; BD Biosciences, #561646) and APC-conjugated anti-CD44 antibody (1:60 for 20 min; BD Biosciences, #559942) by using PE-Cy™7 Mouse IgG2a (1:100 for 20 min; BD Biosciences, #552868) and APC Mouse IgG2b (1:60 for 20 min; BD Biosciences, #555745) as control staining. Stained cells were analyzed by BD FACSCanto II cytometer and data were acquired by BD FACSDiva v8.0.2 software and processed by FlowJo v10.7.1 software (BD Biosciences). ALDEFLUOR assay was carried out using the ALDEFLUOR assay kit (STEMCELL Inc., #101700) according to the manufacturer’s instructions. The ALDH1 inhibitor, diethylaminobenzaldehyde (DEAB), was used as a negative control. The processed cells were evaluated by BD FACSCanto II cytometer and data were acquired by BD FACSDiva v8.0.2 software and analyzed by FlowJo v10.7.1 software (BD Biosciences).