Nkg2c apc

We investigated the immunophenotyping of functionally relevant receptors within NK subpopulations by specific staining and posterior flow cytometry. We incubated purified NK cells (2 × 10⁶ cells/mL) overnight with 100 ng/mL LPS and 10 µg/mL aCpG in the presence or absence of increasing concentrations of ruxolitinib (0.1 µM, 1 µM, 10 µM) at 37°C in complete media. After incubation, we performed immunophenotyping of functionally relevant receptors within the NK subpopulations with TIGIT PE-Vio770, CD69 BV421, TACTILE BV421 (Biolegend), CD56 AlexaFluor770, CD16 APC-Cy7, CD25 FITC, DNAM-1 PE, CXCR4 APC, NKG2D BV421, KIR3DL1 PE-Vio770, KIR2DL2/L3/S2 FITC, KIR3DL2 PE (BD Biosciences), NKp46 FITC, NKp44 PE, NKp30 APC, CD57 FITC, KIR2DS4 PE, NKG2A PE-Vio615, PD-1 FITC, LAG-3 PE, TIM-3 PE-Vio770, KIR2DL4 APC, KIR2DL1 BV421, CD3 Viogreen (Miltenyi Biotec), and NKG2C APC (R&D Systems). We acquired samples in a Navios flow cytometer (Beckman Coulter) and employed FlowJo v10.0.7 software (BD Biosciences) for the data analysis.

We used the following antibodies in this study: NKG2C-APC, NKG2C-PerCp, NKG2A-PE (R&D); CD3-FITC, CD4-APC-Cy7, CD16-PerCP-Cy5.5, CD56-PE-Cy7, CD107a-APC-H7 (BD Biosciences, San Jose, CA, USA); IFN-γ-BV421 (Biolegend, San Diego, CA, USA); CD4-FITC/CD8-PE/CD3-PerCP (BD Biosciences, San Jose, CA, USA).