Anti cd107a pe

In cytotoxic T cells, the cell surface mobilization of the CD107a was assessed as a marker for degranulation of lytic granules. In brief, 5 × 10⁵ gene-transduced CD8⁺ T cells were incubated with 5 × 10⁵ peptide-pulsed T2 cells for 4 h in the presence of 10 µg/ml brefeldin A and 5 µl PE-anti-CD107a (eBioscience). After the incubation, the cells were harvested, washed, and stained with antibody specific for CD8. Data analysis was performed by Flow cytometry.

Single-cell suspensions were pre-incubated with Fc-receptor blocking solution (anti-mouse CD16/32, #14-0161-82, eBioscience, California, USA) for 30 min, and stained with the fluorochrome-conjugated antibodies conjugates at 4˚C for at least 1h. The following mAb were used: FITC-anti-CD4 (#4313007), FITC-anti-CD8α (#4271604), FITC-anti-CD11c (#E00155-1631), PE-anti-CD69 (#E01333-1634), PE-anti-CD8α (#E01038-1633), PE-anti-CD86 (#E01369-1634), PE-anti-CD107a (# E01430-1632), PE-anti-B7H1 (#4276913), PE-anti-CD49d (#E01278-1635), PE-anti-CXCR5 (#E16203-104), Percp-Cy5.5-anti-CD3e (#4304569), Percp-Cy5.5-anti-FOXP3 (#E08398-1634), APC-anti-CD25 (#E07106-1634), APC-anti-CD80 (#4329685), APC-anti-PD-1 (#4344425), eFluor 450-anti-Ki-67 (#1928649), eFluor 450-mouse hematopoietic lineage (#2324728) from eBioscience (California, USA); PE-anti-MHC-II (#107608), Percp-Cy5.5-anti-CD11a (#101124), BV421-anti-LAG3 (#125221), PE-Cy7-anti-TIGIT (#142107), AF700-anti-mouse CD317 (#127037), APC-Cy7-anti-CD107a (#121615) from Biolegend (San Diego, USA); PE-CF594-anti-CD8α (#562283), BV510-anti-CD11a (#563669) from BD Bioscience (Bedford, USA). All data were acquired on BD FACS Calibur, BD FACS Celesta or BD FACS Aria III flow cytometer and analyzed with FlowJo software (BD Life Sciences, Franklin Lakes, NJ, USA).

After Fc receptors blocking (BD Biosciences, USA), an appropriate concentration of the fluorescence-labeled antibody was used for the staining of surface antigens at 4 °C for 30 min in dark place. Fluorochrome-conjugated monoclonal antibodies of cellular markers: PercpCy5.5-anti-CD3, FITC-anti-IL-17A, PE-anti-CD4, PE-Cy7-anti-NK1.1 (BD Bioscience, USA). FITC-anti-CD69, FITC-anti-IFN-γ; PE-anti-FasL, PE-anti-TRAIL, PE-anti-CD107a, and APC-anti-NKG2D (eBioscience, San Diego, CA). The intracellular cytokine staining, including INF-γ and IL-17A, was using Mouse Intracellular Cytokine Staining Starter Kit (BD Biosciences, USA), according to the kit’s instructions. Samples were measured by a BD Accuri C6 plus flow cytometer (BD Biosciences, USA), and the data were managed using BD Accuri C6 plus analysis (BD Biosciences, USA).