Human nk cell isolation kit

Human blood samples from normal healthy donors were drawn for research purposes under a protocol approved by the institutional review board with informed consent. PBMCs were isolated by density gradient centrifugation using lymphocyte separation medium (MP Biomedicals, Solon, OH). Human NK cells were purified from PBMCs by negative selection using a human NK cell isolation kit (StemCell Technologies) as described (33 (link)). These cells were 97–99% CD3-CD56+ as assessed by flow cytometry. The human NK cell line NKL was cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, and 200 U/ml recombinant IL-2 (rIL-2) (Roche). K562, 721.221, and KCL-22 cells were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% FBS and 2 mM L-glutamine. YAC-1, RMA, and RMA-s cells were cultured in RPMI1640 supplemented with 5% FBS and 2 mM L-glutamine. B16F10 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 2 mM L-glutamine. In some experiments, PBMCs were activated with 200 U/ml rIL-2 for 24 h. Male C57BL/6 mice were used at 6–7 weeks old. Murine NK cells were purified from splenocytes by negative selection using a mouse NK cell isolation kit (StemCell Technologies) according to the manufacturer's instructions.

Blood and tumour biopsies were collected during surgical resection. Tumour biopsies were enzymatically digested with collagenase type IV as previously^{16 (link),27 (link)}. Adipose tissue conditioned media (ACM) and tumour tissue conditioned media (TCM) were prepared as previously described^{16 (link),28 (link)}. PBMC were prepared by density gradient centrifugation. Non-cancer healthy control primary NK cells were isolated from PBMC by magnetic cell sorting using human NK cell isolation kit (Stemcell) according to manufacturer’s instructions. NK cells were expanded using MACS media (Miltenyi Biotech) supplemented with human AB serum (Sigma) and IL-2 (Peprotech) as per manufacturer’s instructions.

Natural killer cells were harvested by using human NK cell isolation kit (#17955, Stem Cell Technologies). To assess the efficiency of NK cell sorting, enriched cells were analyzed by flow cytometry. Total RNA was extracted from NK cells using RNA‐quick purification kit (#RN001, Yi Shan Biotechnology), followed by RNA quality assay (Nanodrop, Thermo Scientific) and first‐strand cDNA synthesis (#K1622, Thermo Scientific). Amplification of cDNA was performed on ABI‐7500 system (Applied Biosystems). Primer sequences for RT‐qPCR were shown in Table S2. The results of gene expression were calculated using the 2‐ΔΔCT method.