Perforin fitc

MHC class I tetramer was prepared in-house. Surface staining of T cells was achieved by addition of tetramer to whole blood, incubation for 10 minutes at room temperature, followed by addition of antibody co-stains for 20 minutes. Whole blood was preferred to thawed PBMCs for tetramer labelling due to greater consistency and signal intensity. Following lysis of erythrocytes using BD FACS Lysing solution (BD Biosciences), cells were either fixed using 2% formaldehyde (v/v) or permeabilized using the BD Cytofix/Cytoperm kit for intra-cellular staining. The following antibodies were used for surface and intra-cellular staining: CD3-PerCP, CD8-Horizon V500, CD8-APCH7, Ki-67-FITC, Bcl-2-PE, HLA-DR-Horizon V450, HLA-DR-PerCP, Perforin-FITC, Granzyme B-Horizon V450, CCR7-PE, CD27-Horizon V450, CD27-FITC, CD28-PECy7, CD28-PE (all BD Biosciences) and CD38-PECy7 (eBioscience), Granzyme B-PE (Caltag), Granzyme K-PE (Santa Cruz), CD45RA-FITC (Beckman Coulter), PD-1-PE and 2B4-PerCPCy5.5 (Biolegend). Intra-cellular cytokine staining with IFN-γ-FITC, TNF-α-APC, and IL-2-PerCpCy5.5 (all BD Biosciences) was undertaken after in vitro stimulation of PBMCs using peptide for 6 hours. Flow cytometry analysis was performed BD LSRII and BD FACSCanto flow cytometers. Flow cytometry data were analyzed using FlowJo software.

The anti-human mAbs have been used: CD3 PE (from Beckman Coulter), CD3 PErCP, CD11b PE, CD14 APC, CD56 PE, CD56 APC, CD73 PE, CD107a FITC, Granzyme A FITC, Perforin FITC, pJNK PE (from BD Biosciences), HLA-DR FITC (Dako), CD44 FITC, CD105 FITC, Granzyme B PE, Granzyme K Alexa647 (Immunotools), CD90 APC, COX2 FITC (Cayman chemicals). Each flow cytometric analysis was controlled with isotype-matched mAbs. All flow cytometry-based experiments were performed on FACS Calibur using Cell-Quest Pro Software. Offline data analysis was done on Summit 5.1 software.

For assessment of intracellular perforin production, NK cells were incubated with Ghost violet 540 cell viability dye (TONBO Biosciences) and CD56-PerCP (BioLegend). After washing with MACS buffer, cells were then fixed and permeabilized, following instructions in the Intracellular Fixation and Permeabilization Buffer Set (Thermo Fisher). Cells were then incubated overnight with Perforin-FITC (BD Pharmingen), and analyzed by flow cytometry.