Nk rosettesep

PB-NK cells were harvested from healthy adult volunteers (lab and healthcare workers in the Texas Medical Center), or purchased from the Gulf Coast Regional Blood Center for more extensive experiments. Volunteers are not age or sex matched. Blood was treated with NK Rosette Sep (Stem Cell Technologies) as per manufacturer’s instructions then separated by density centrifugation as described (13 (link)). Cells were washed twice, counted, and either further enriched for NK cells before sorting, sorted directly, or used as controls for flow cytometry or ⁵¹Cr release assays. For total PBMC isolation, peripheral blood was immediately diluted 1:1 with phosphate buffered saline (PBS) and layered over Ficoll-Pacque PLUS (GE Healthcare), centrifuged, washed, and counted for use as controls.

Leucopacks were treated using a negative enrichment antibody mixture (NK RosetteSep, Stem Cell Technologies). Enriched cells were then flow cytometry sorted in order to isolate CD56^bright (CD3⁻ CD56^bright CD16⁻), CD56^dim (CD3⁻ CD56^dim CD16⁺) subsets, as well as an intermediate population which is CD3⁻ CD56^bright CD16^low. Cells were washed 3-4 times with PBS and cell pellets stored at -80C. RNA was isolated using the RNeasy Plus Mini Kit (Qiagen). All isolated RNAs showed an RNA Integrity Number of >9.3 when tested by Agilent Bioanalyzer. First strand cDNA was synthesized from 100-200ng total RNA using RT^{2 (link)} First Strand Kit (Qiagen) and PCR reactions were performed with RT² SYBR Green Mastermix (Qiagen) in triplicate. The PCR reaction profile consisted of 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, and 1 min at 60 °C in BioRad CFX96 or Stratagene MX3000p thermocyclers. Primers were derived from custom designed RT² Profiler PCR Arrays (SuperArray/Qiagen). Samples with multiple melting-curve peaks were not included in analyses.

Human pNK cells were enriched from leukopacks using a negative enrichment Ab cocktail (NK RosetteSep, StemCell Technologies, Vancouver, BC, Canada) following the manufacturer’s instructions. For microarray analysis CD3^- CD56^Bright CD16^- pNK and CD3^- CD56^Dim CD16⁺ pNK cells were further purified by FACS sorting. Alternatively enriched pNK cells were seeded at 1 X 10⁶ cells/ml in IL-15 complete media (RPMI 1640 media containing 10% FCS, 1 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, nonessential amino acids, and 55 nM 2-ME [all from Life Technologies, Grand Island, NY] with the addition of 10 ng/ml recombinant human IL-15 [PeproTech, Rocky Hill, NJ]) and maintained in culture in humidified incubators at 37°C with 21% O₂ and 5% CO₂ for 7 days (for injection at gd9) or 10 days (for injection at gd12) to be used as controls in animal experiments.