Complete blood counts were determined using an automated hematology analyzer in duplicate (Sysmex XN-330, Sysmex Co., Kobe, Japan). Immediately after complete blood counting, peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation as previously described (Stem Cell Technologies, Vancouver, Canada) (19 (link)). After isolation, PBMCs were counted using a flow cytometer (BD Accuri C6, Ann Arbor, MI) and an automated cell counter (Countess 3 FL;ThermoFisher Scientific Inc.) Then, according to the manufacturer’s instructions, NK cells were isolated by negative selection using magnetic-activated cell sorting (MACS) separation beads (Miltenyi Biotec, Auburn, CA). Negatively-sorted NK cells were washed at 400g for 10min before resuspending with RPMI-1640. The purity of cells after separation was confirmed using anti-CD3, and anti-CD56 antibodies, to ensure a minimum purity of 90% NK cells were achieved.
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Isolation and Purification of NK Cells
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Corresponding Organization : Pennington Biomedical Research Center
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Variable analysis
independent variables
- Isolation of peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation
- Isolation of natural killer (NK) cells by negative selection using magnetic-activated cell sorting (MACS)
- Complete blood counts
- PBMC count using flow cytometry and automated cell counter
- Purity of NK cells after separation (minimum 90%)
- Automated hematology analyzer (Sysmex XN-330)
- Flow cytometer (BD Accuri C6)
- Automated cell counter (Countess 3 FL)
- MACS separation beads (Miltenyi Biotec)
- RPMI-1640 medium
- No positive or negative controls were explicitly mentioned in the provided information.
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