Dnam 1 pe

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.

MM cells were incubated with 0, 5, 10, and 20 nM bortezomib for 12 h and MM cells were incubated with 10 nM bortezomib for 12, 24, and 36 h. Cells were then stained with Nectin-2-PE (BD Biosciences), PVR-PE, MICA-PE, and MICB-APC (R&D Systems, Minneapolis, MN, USA). Purified induced NK and γδ T cells were stained with CD56-FITC (BD Biosciences) or Vγ9-FITC, CD3-PerCp (BD Biosciences), NKG2D-APC (BD Biosciences), and DNAM-1-PE (BD Biosciences). Appropriate isotype-matched antibodies were used as controls. Data were analyzed using a BD FACSCalibur (BD Biosciences) with Cell Quest Pro software and final analysis was performed using FlowJo software (Tree Star Inc., Ashland, OR, USA).

Expanded γδ T cells treated with 0.5 µM DAC for 48 h were stained with DNAM-1-PE (559789), NKG2D-APC (558071), Vγ9-FITC (555732), KIR2DL2/3 (CD158b)-PE (559785), CD3-PerCP (347344), KIR2DL1 (CD158a)-PE (556063) (BD Biosciences), CD279-APC (329908) (BioLegend, San Diego, CA, USA), KIR2DS4 (CD158i)-APC (FAB1847A), and KIR3DL1 (CD158e1)-APC (FAB1225A) (R&D Systems, Minneapolis, MN, USA). Appropriate isotype-matched antibodies (Abs) were used as controls. Data were analyzed by flow cytometry.