Live dead fixable near ir

NK cell degranulation upon co-incubation with target cells was determined by the expression of CD107a on the cell surface, which serves as a surrogate marker for NK cell degranulation [58 (link)]. In brief, overnight cultured NK cells enriched with the EasySep human NK cell enrichment kit (StemCell Technologies) from PBMCs from KIR2DL5A*001-positive donors or donors lacking KIR2DL5 genetically were co-cultured with CD155-expressing (CD155-transduced 721.221) or CD155-nonexpressing (721.221) target cells at an effector to target ratio of 1:2 in 200 μl complete R10 for 4 h at 37°C. During co-incubation, each well contained 2 μl anti-CD107a (clone LAMP-1, Biolegend) and 25μl/ml Brefeldin A. Cells were subsequently stained with LIVE/DEAD Fixable Near-IR and with the following antibodies: anti-CD3-BUV395 (clone UCHT1, BD), anti-CD16-PE-Cy7 (clone 3G8, Biolegend), anti-CD56-BV785 (clone NCAM16.1, BD), anti-KIR2DL5-PE (clone UP-R1, Biolegend) for 15 min at RT and fixed with CellFix (BD) before flow cytometric acquisition.

CD4⁺ and CD8⁺ lymphocyte activation was analyzed on thawed and over-night rested PBMCs by flow cytometry. Cells were stained for 30 min at 4°C with LIVE/DEAD Fixable NEAR-IR in order to exclude dead cells, and with the following fluorochrome-conjugated antibodies (all of them obtained from BD Biosciences, San Jose/California, USA): anti-HLA-DR-FITC, anti-CD4-PerCP, anti-CD38-APC, anti-CD3-PeCy7 and anti-CD8-PE.
Data acquisition and analysis was performed using the BD FACSDiva software. Initial gating was performed on living lymphocytes followed by gating on CD3⁺CD4⁺ or CD3⁺CD8⁺ events. Isotype-matched FITC- and APC-conjugated non-specific antibodies were used in each sample in order to accurately set HLA-DR and CD38 negative populations.