Cd56 apc alexa fluor 750

Peripheral blood mononuclear cells (PBMC) from CLL patients and HC were isolated and cryopreserved as described earlier.^{25 (link)} To enrich for NK cells, samples from CLL patients were CD19-depleted using CD19 immunomagnetic microbeads (Miltenyi Biotec, Bergish Gladbach, Germany). Afterwards, PBMC were washed with ice-cold phosphate-buffered saline containing 0.5% bovine serum albumin (PBA) and stained for 30 minutes at 4°C with saturating amounts of CD56 APC-Alexa Fluor 750, CD16 ECD, CD158 (a,h) APC, CD158 (e1,e2) APC, CD158 (b1, b2, j) APC, p75 PE, NKG2A PE-Cy7, BTLA PC7, CD160 PE, ILT2 APC (Beckman Coulter, Brea, CA, USA), KLRG1 Alexa Fluor 488, NKG2D Pe-Cy7 (ThermoFisher Scientific), CD3 FITC, NKp30 APC, CD57 PerCP-Cy5.5 (Biolegend, San Diego, CA, USA), CD3 PE (BD Biosciences, Franklin Lakes, NJ, USA) and NKG2C Alexa Fluor 700 (R&D systems, Minneapolis, MN, USA). Cells were washed twice with PBA, resuspended, and analyzed on a BD FACSCanto flow cytometer. Data analysis was performed using Flowjo Mac Version 10. Gating strategy can be found in supplemental Figure 4 (Supplemental Digital Content).