The following antibodies were used to stain peripheral blood mononuclear cell (PBMC) suspensions: fluorescein isothiocyanate (FITC)-CD3, FITC-CD4, peridinin-chlorophyll-protein (PerCP/cyanine 5.5-CD16, phycoerythrin (PE)-CD19, PE-CD56, adenomatous polyposis coli (APC)-CD158 (KIR2D L1/S1/S3/S5), APC-CD158e1 (KIR3DL1), APC-CD159a (NKG2A), APC-CD226 (DNAM-1), APC-CD244 (2B4), APC-CD314 (NKG2D), and Alexa Fluor (AF)647-CD337 (NKp30) (BioLegend, San Diego, CA, USA), eFluor660-CD107a (eBioscience, San Diego, CA, USA), and CD8 (BD Biosciences, Franklin Lakes, NJ, USA). The subpopulation of circulating NK cells was quantified and analyzed using flow cytometry, as described previously.32 (link)
Briefly, 5 × 105 PBMCs after red blood cell lysis were collected for incubation with antibodies for 30 minutes at 4°C, washed with phosphate-buffered saline and fixed with 4% paraformaldehyde for 20 minutes. The cells were then washed twice with phosphate-buffered saline and analyzed using FACSCalibur (BD Biosciences). Data were analyzed using Cell Quest Pro (FlowJo, LLC, Ashland, OR, USA). Human plasma IL-6 and TNF-α levels were measured with commercially available Quantikine Enzyme-Linked Immunosorbent Assay Kits (R&D Systems, Inc., Minneapolis, MN, USA) in accordance with the manufacturer’s instructions.
Briefly, 5 × 105 PBMCs after red blood cell lysis were collected for incubation with antibodies for 30 minutes at 4°C, washed with phosphate-buffered saline and fixed with 4% paraformaldehyde for 20 minutes. The cells were then washed twice with phosphate-buffered saline and analyzed using FACSCalibur (BD Biosciences). Data were analyzed using Cell Quest Pro (FlowJo, LLC, Ashland, OR, USA). Human plasma IL-6 and TNF-α levels were measured with commercially available Quantikine Enzyme-Linked Immunosorbent Assay Kits (R&D Systems, Inc., Minneapolis, MN, USA) in accordance with the manufacturer’s instructions.
