Anti cd107 pe

NK cell degranulation assays were performed as elsewhere described (37 (link), 38 (link)). Briefly, PBMCs (1x10⁶/ml) were incubated with P815 cells (2x10⁶/ml) in a total volume of 200 μl in a 96 well plate. P815 cells were supplemented with 5mg/ml of anti-CD16 mAb (Clone 3G8, eBiosciences). After 3 hours of incubation at 37°C, cells were recovered and stained using following antibodies: anti-CD3 FITC, anti-CD56 APC, and anti-CD107 PE (Biolegend, clone H4A3). GolgiStop was not included in these assays. Cells were acquired on FACSCanto II (BD bioscience) and analyses with the use of FlowJo 7.6.5 software (Tree Star, Ashland, OR). Gates were set to exclude CD3+ lymphocytes. Thereafter, the percentage of cells positive for CD107a was obtained after gating in CD3-CD56+ lymphocytes. The basal percentages for CD107a were obtained from PBMCs incubated with P815 cells with no agonist mAb. Degranulation was represented as the fold increase for CD107a, which is the difference between the percentage of NK cells expressing CD107a at surface after stimulation with P815 cells supplemented with agonist mAb, and the percentage of NK cells expressing CD107a at NK cell surface after incubation with P815 cells with no agonist mAb. NK cell degranulation assays were performed in 32 acute leukemia patients and 10 age-matched healthy controls.

NK cell degranulation assays were performed as previously described [32 (link)–34 (link)]. Briefly, PBMCs (1x10⁶/ml) were incubated with K562 cells (2x10⁶/ml) in a total volume of 200 ul in a 96 well plate. After 4 hours of incubation at 37°C, cells were recovered and stained using following antobodies: anti-CD3 FITC, anti-CD56 APC, and anti-CD107 PE (Biolegend, clone H4A3). GolgiStop was not included in these assays. Cells were acquired on FACSCanto II (BD bioscience) and analyses with the use of FlowJo 7.6.5 software (Tree Star, Ashland, OR). Gates were set to exclude CD3⁺ lymphocytes. Thereafter, the percentage of cells positive for CD107a was obtained after gating in CD3^-CD56⁺ lymphocytes. The basal percentages for CD107a were obtained from PBMCs incubated alone. Degranulation was represented as ΔCD107a, which is the difference between the percentage of NK cells expressing surface CD107a after K562 stimulation and the percentage of NK cells expressing surface CD107a after incubation with medium alone. NK cell degranulation assays were performed in 41 acute leukemia patients and 14 age-matched healthy controls.

NK cell degranulation assays were performed as previously described. Briefly, peripheral blood mononuclear cells (PBMCs, 1x10⁶/ml) were incubated with K562 cells (2x10⁶/ml) in a total volume of 200 μl in a 96-well plate. After 4 hours of incubation at 37°C, cells were recovered and stained using the following antibodies: anti-CD3 FITC, anti-CD56 APC, and anti-CD107 PE (Biolegend, clone H4A3). GolgiStop was not included in these assays.
Cells were acquired on FACSCanto II (BD bioscience) and analyzed with FlowJo 7.6.5 software (Tree Star, Ashland, OR). Gates were set to exclude CD3+ lymphocytes. Thereafter, the percentage of cells positive for CD107a was obtained after gating in CD3-CD56+ lymphocytes. The basal percentages for CD107a were obtained from PBMCs incubated alone. Degranulation was represented as CD107a, which is the difference between the percentage of NK cells expressing surface CD107a after K562 stimulation, and the percentage of NK cells expressing surface CD107a after incubation with medium alone.