The antibodies used for CTL phenotyping (anti-CD3-ECD, CD4-PC5, CD8-PC7, PD-1-PE, CTLA-4PE, PD-L1 FITC; IgG1-FITC, IgG1-PE) were all obtained from BD Biosciences. To investigate competitive binding of staining antibody with blocking PD-1 mAb, flow cytometric analysis was performed using two different clones of staining antibody after 48 hrs: primary PD-1 (MIH4), secondary IgG1 FITC and PD-1 conjugated with PE.
Positive staining for PD-1 and PD-L1 was determined by comparing with the respective isotype control. The cell suspensions were incubated with mAb for 30 min at 4°C and then washed twice in PBS. PD-1 mAb-treated co-cultures were incubated with an anti-CD107a antibody and degranulation assay was performed.29 (link) The cells were acquired on an FC 500 (Beckman Coulter) and analyzed by the CXP analysis software.
Positive staining for PD-1 and PD-L1 was determined by comparing with the respective isotype control. The cell suspensions were incubated with mAb for 30 min at 4°C and then washed twice in PBS. PD-1 mAb-treated co-cultures were incubated with an anti-CD107a antibody and degranulation assay was performed.29 (link) The cells were acquired on an FC 500 (Beckman Coulter) and analyzed by the CXP analysis software.
