Easysep negative human nk cell isolation kit

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by centrifugation over a density gradient medium (Ficoll-Paque Premium; GE Healthcare, Uppsala, Sweden) to separate granulocytes as previously described [3 (link), 43 (link)]. PBMCs were stained with trypan blue stain (Invitrogen Life Technologies, Carlsbad, CA, USA) to determine total cell count and cell viability and adjusted to a final concentration of 5 × 10⁷ cells/ml. NK cells were isolated from PBMCs using an EasySep Negative Human NK Cell Isolation Kit (Stemcell Technologies, Vancouver, BC, Canada). NK cell purity was measured following staining with CD56-Pe-Cy7 (0.25 µg/5 µl) and CD3-APC-H7 (0.5 µg/5 µl) antibodies (Beckon Dickinson [BD] Bioscience, Miami, FL, USA) for 20 min (min) at room temperature in the dark and analysed using a LSR-Fortessa X20 flow cytometer (BD Biosciences, Miami, FL, USA). NK cell purity was 92.46 ± 2.375 for HCs and 93.58 ± 0.9282 for ME/CFS, respectively (Additional file 1).

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by centrifugation over a density gradient medium (Ficoll-Paque Premium; GE Healthcare, Uppsala, Sweden) to separate granulocytes as previously described [27 (link), 28 (link)]. PBMCs were stained with trypan blue stain (Invitrogen, Carlsbad, CA) to determine total cell count and cell viability and adjusted to a final concentration of 5 × 10⁷ cells/ml. NK cells were isolated from PBMCs using an EasySep Negative Human NK Cell Isolation Kit (Stemcell Technologies, Vancouver, BC, Canada) as previously described [27 (link), 28 (link)].

A total of 85 ml of whole blood was collected in ethylendiaminetetraacetic acid (EDTA) tubes between 8:00 am and 12:00 am. Routine full blood analysis was performed within 4h of collection for red blood cell count, white blood cell count and granulocyte cell count.
Peripheral blood mononuclear cells (PBMCs) were isolated from 80 ml of whole blood by centrifugation over a density gradient medium (Ficoll-Paque Premium; GE Healthcare, Uppsala, Sweden) as previously described (Brenu et al., 2011 (link); Munoz & Leff, 2006 (link)). PBMCs were stained with trypan blue (Invitrogen, Carlsband, CA, USA) to determine cell count and cell viability. PBMCs were adjusted to a final concentration of 5 × 10⁷ cells/ml for NK cell isolation.
NK cells were isolated by immunomagnetic selection using an EasySep Negative Human NK Cell Isolation Kit (Stem Cell Technologies, Vancouver, BC, Canada). NK Cell purification was determined using flow cytometry. NK cells were incubated for 20 min at room temperature in the presence of CD56 FITC (0.25 μg/5 μl) and CD3 PE Cy7 (0.25 μg/20 μl) monoclonal antibodies (BD Bioscience, San Jose, CA, USA) as previously described (Nguyen et al., 2017 (link)).