Pe conjugated anti nkg2c

To evaluate, by multicolor flow cytometry, the expression of NKG2C, NKG2A, CD57, NKG2D, DNAM-1, KIRs, and PD-1 on NK cells, thawed PBMCs were stained with the LIVE/DEAD™ Fixable Near-IR Dead Cell stain dye (from Invitrogen, Waltham, MA, USA) for 15 min at room temperature (RT). Before fixing for 20 min at RT with 1% PFA, cells were stained for 20 min at 4 °C with PerCP-Cy5.5-conjugated anti-CD56, BV510-conjugated anti-CD16, Alexa Fluor 700-conjugated anti-CD3, BV605-conjugated anti-CD14, BV650-conjugated anti-CD19, PE-CF594-conjugated anti-NKG2D, BV786-conjugated anti-DNAM-1, BV421-conjugated anti-PD-1 (all from BD Biosciences, San Jose, CA, USA), PE-conjugated anti-NKG2C, FITC-conjugated anti-NKG2A (both from Miltenyi Biotec, Bergisch Gladbach, DE), PE-Cy7-conjugated anti-CD57 (from Life Technologies, Carlsbad, CA, USA), APC-conjugated anti-CD158 (KIR2DL1/S1/S3/S5), and anti-CD158b/j (KIR2DL2/L3) (both from BioLegend, San Diego, CA, USA) mAbs. Stained samples were acquired at CytoFLEX Flow Cytometer from Beckman Coulter Life Sciences and all results were analyzed using FlowJo version X.0.7 software.

To determine, by multicolor flow cytometry, CD107a (LAMP-1) surface mobilization on NK cells, thawed PBMCs were co-cultured (1:1) with human erythroleukemia K562 target cells for 4 h at 37 °C in the presence of FITC-conjugated anti-CD107a (from BD Pharmingen, San Diego, CA, USA) mAb. After the first hour, 0.66 μL/mL of BD GolgiStop (protein transport inhibitor containing monensin) was added. At the end of stimulation, cells were washed with PBS + 1% FCS + 5mM EDTA to promote conjugate disruption and stained with the LIVE/DEAD™ Fixable Near-IR Dead Cell stain dye (from Invitrogen, Waltham, MA, USA) for 15 min at RT. Before fixing for 20 min at RT with 1% PFA, cells were stained for 20 min at 4 °C with BV510-conjugated anti-CD16, PE-CF594-conjugated anti-CD3, BV605-conjugated anti-CD14, APC-conjugated anti-CD57, BV786-conjugated anti-DNAM-1, BV421-conjugated anti-PD-1 (all from BD Biosciences, San Jose, CA, USA), PerCP-conjugated anti-CD56 (from Invitrogen, Waltham, MA, USA), APC Alexa Fluor 750-conjugated anti-CD19 (from Life Technologies, Carlsbad, CA, USA), PE-conjugated anti-NKG2C (from Miltenyi Biotec, Bergisch Gladbach, DE, USA), and PE-Cy7-conjugated anti-NKp46 (from BioLegend, San Diego, CA, USA) mAbs. Stained samples were acquired using a CytoFLEX Flow Cytometer from Beckman Coulter Life Sciences and all results were analyzed using FlowJo version X.0.7 software.