Pe conjugated anti cd140a

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.