Anti human cd3 percp cy5

For surface staining, PBMCs from liquid nitrogen were thawed and washed twice in phosphate buffered saline containing 1% fetal bovine serum (staining buffer). Cells were incubated with directly conjugated monoclonal antibodies for 30 min at 4 °C. The cells were then washed and resuspended in staining buffer before flow cytometric analysis. The monoclonal antibodies used were anti-human CD3-PerCp-Cy5.5 or CD3-BV421, CD4-FITC or CD4-V500, CD8-APC-H7, CD45RA-PE-Cy7, CCR7-PerCp-Cy5.5 (BD Biosciences, San Diego, CA, USA), PD-1-PE, TIM-3-FITC, 2B4-APC, BTLA-BV421, CD160-PE-Cy7 (BioLegend, San Diego, CA, USA) and LAG-3-AF700 (R&D Systems, Minneapolis, MN, USA) antibodies, and corresponding isotype controls. Data acquisition was performed on a LSR Fortessa flow cytometer (BD Biosciences) and data analysis was performed using FlowJo Software (Tree Star, Ashland, OR, USA).

Flow cytometry was performed on a BD FACS Celesta flow cytometer. Data were graphed using FlowJo 7.6. In all cases, cells were washed three times with PBS and then incubated with antibodies for 30 min at 37°C in the dark. Before analysis, cells were washed once and then resuspended in 500 μl PBS. Phenotypic characterization of T cells was determined by directly staining with anti-human CD3-PerCP-cy5.5 (BD Pharmingen, 560835), anti-human CD4-FITC (eBioscience, E10526-1634), anti-human-CD8-APC (eBioscience, 4280528). The CAR-encoded ZsGreen fluorescent protein can be detected by flow cytometry, and the transduction efficiency of T cells was shown by detecting GFP fluorescence on CAR-T cells. The expression of c-Met and PD-L1 on tumor cells was examined by incubating with APC-conjugated anti-c-Met antibody (Sino Biological, 10692-R243-A) and FITC-conjugated anti-PD-L1 antibody (Sino Biological, 10084-R611-F).