Annexin v 7 aad assay

To measure apoptosis, we utilized the Annexin-V/7-AAD assay BD Bioscience# 559763; San Jose, CA 95131-USA) which detects both early and late events in apoptosis. Floating and attached cells were stained following the kit guidelines to analyze apoptosis and were evaluated using an LSRIIB-FACS analyzer. When used together, 7-AAD and Annexin-V provides a simple staining assay to monitor apoptosis by flow cytometry that allows one to differentiate between 1) intact cells, 2) cells in early apoptosis, which only stain positive for Annexin-V, and 3) cells in later apoptosis, which only stain for 7-AAD.

NK cells were isolated from PB samples obtained from healthy volunteers. Undiluted PB was overlaid on a Ficoll density gradient and centrifuged at 200× g for 20 min without break. The platelet-enriched fraction was collected and stored at RT. The mononuclear cells were collected, and NK cells were isolated by negative depletion using the EasySep human NK cell isolation kit and EasySep magnet (Stemcell Technologies, Vancouver, BC, Canada), according to the manufacturer’s instructions. Isolated NK cells were cultured for 7 days in NK MACS medium (Miltenyi Biotech GmbH; Bergisch Gladbach, Germany) supplemented with human serum, IL-2 (500 U/mL,) and IL-15 (140 U/mL), both from Stemcell Technologies. Media was replenished every 3 days. MM cells were incubated with/without platelets and after incubation, cells were cocultured with/without platelet-autologous NK cells at different ratios for 5 h. Cell death was measured by flow cytometry using the Annexin V/7AAD assay (BD Biosciences).