Cd19 apccy7

PBMCs isolated from HDLS donor blood were seeded in round bottomed 96‐well plates in Roswell Park Memorial Institute (RPMI) media with 10% FBS at 4 × 10⁵ cells per well and incubated in a 5% CO₂ incubator at 37°C overnight. Cells were centrifuged at 300 g for 5 min and blocked for 10 min with 50 μl 10% human AB serum, 1% FBS in PBS. Blocked PBMCs were incubated in Live/Dead Aqua (1:500; Life technologies (Fisher/Invitrogen) L34957 ) for 30 min, washed in PBS and incubated in fluorochrome‐labelled primary antibody diluted 1:10 in PBS, 1% FBS for 20 min at 4°C. (Lin FITC (BioLegend 348801), CD3 PerCP (Miltenyi 130‐100‐458), CD19 APCCy7 (Miltenyi 130‐098‐073), CD14 PacBlue (Miltenyi 130‐098‐058). Cells were washed twice with PBS 1% FBS and analysed by flow cytometry immediately. Gating during analysis was based on fluorescence minus one controls. Flow cytometry was carried out on a BD LSRFortessa cell analyser and analysed using FlowJo software (Tree Star).

NK cells were enriched from freshly isolated PBMCs using an EasySep Human NK Cell Enrichment Kit (Stemcell, Vancouver, Canada) as per the manufacturer's guidelines and sorted using a BD FACS Aria-Fusion. NK cells were labeled with CD3-AF700, CD14-APC-Cy7, CD19-APC-Cy7, NKG2A-APC (Miltenyi Biotec, Bergisch Gladbach, Germany–REA110), LIVE/DEAD fixable near-IR dye and a combination of KIR2DL1/KIR2DS5-PE, KIR2DL2/S2/L3-PE (BioLegend–DX27), and KIR3DL1-PE (BioLegend–DX9) depending on specific donor genotype for 20 min at 4°C in the dark. Cells were sorted to select for NKG2A-educated NK cells and uneducated NK cells while excluding KIR-educated NK cells and were subsequently incubated overnight in RPMI containing 10% (v/v) FBS with 1 ng/mL IL-15. NK cells were washed then resuspended in calcium buffer (HBSS + 1 mM CaCl2 + 0.5 mM MgCl2 + 10 mM HEPES + 0.1% FBS) and stained with 4 μM Fura2 (Life Technologies, CA, USA) for 30 min at 37°C. Cells were resuspended in calcium buffer then observed under a fluorescent microscope. Biotinylated anti-NKp46 (BioLegend–9E2) and anti-2B4 (BioLegend–C1.7) antibodies were added after 3 min then streptavidin (BioLegend) after a further 2 min. Thapsigargin (Merck Millipore, MA, USA) was added at 17 min. The 340/380 ratio of Fura2 was recorded to measure intracellular calcium release by cells over time at 37°C using an incubator chamber.