Pe hamster anti mouse ctla

PBMCs (1x10⁶ cells in each tube) were stained with Alexa Fluor 488 hamster anti-mouse CD3e (BD Biosciences, clone 145-2C11), Pe-Cy7 rat anti-mouse CD4 (BD Biosciences, clone GK1.5), APC-H7 rat anti-mouse CD8a (BD Biosciences, clone 53–6.7), APC hamster anti-mouse PD-1 (BD Biosciences, clone J43), and PE hamster anti-mouse CTLA (BD Biosciences, clone UC10-4F10-11), Fixable Viability Stain 510 (BD Biosciences, cloneR35-95). Corresponding isotype control for each antibody was prepared for appropriate setting of gates during multicolor flow cytometry analysis. All antibodies were pre-titrated for optimal working concentration. Data were acquired on an 8-color FACSCanto II immunocytometry system (BD Biosciences) with BD FACSDiva software (BD Bioscience). Data were exported from BD FACSDiva and analyzed using Flowjo software version 10 (Tree Star, Oregon, USA).

In each tube, 1x10^6 PBMCs were stained with Fixable Viability Stain 510 (BD Biosciences, cloneR35-95), Alexa Fluor 488 hamster anti-mouse CD3e (BD Biosciences, clone 145-2C11), Pe-Cy7 rat anti-mouse CD4 (BD Biosciences, clone GK1.5), APC-H7 rat anti-mouse CD8a (BD Biosciences, clone 53–6.7), APC hamster anti-mouse PD-1 (BD Biosciences, clone J43), and PE hamster anti-mouse CTLA (BD Biosciences, clone UC10-4F10-11). Respective isotype control for each antibody was used to place proper gate for backgrounds. All antibodies were pre-titrated for optimal working concentration. Data were acquired on 8-color FACS Canto II (BD Biosciences) immunocytometry system with FACS Diva software (BD Bioscience). Data were analyzed using Flowjo software version 10 (Tree Star, Oregon, USA).