Granzyme b fitc clone gb11

Analysis of the expression of cell surface and intracellular proteins in purified NK cells and in human carcinoma cell lines was performed by flow cytometry. Cells (1.0 × 10^{6 (link)}) were incubated with 1 μL per test of LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA) in 1 × phosphate buffered saline (PBS) for 30 min at 4°C to accomplish live versus dead cell discrimination. Cells were then centrifuged, washed twice with cold PBS, and then stained with primary antihuman mAbs in 1 × PBS +1% BSA (Teknova, Hollister, CA) for 30 min at 4°C. Binding of NEO-201 to human carcinoma cell lines was detected by Pacific Blue-conjugated NEO-201 antibody (BioLegend, San Diego, CA). To detect the NK markers modulated by ALT-803, purified NK cells were labeled with following antibodies: CD56-PE (clone 5.1H11), CD16-PerCP-Cy5.5 (clone 3G8), Tim-3-PE-Cy7 (clone F38–2E2), NKG2D-BV421 (clone 1D11), CD107a-APC-Cy7 (clone H4A3), Granzyme B-FITC (clone GB11), PD-1-APC (clone EH12.2H7), and CD158d-APC (clone mAb 33) (BioLegend). After staining, cells were washed twice with cold PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA). Analysis of cellular fluorescence was performed using BD FACSuite software (BD Biosciences). Positivity was determined by using fluorescence-minus-one controls.