Anti nkg2d clone 149810

Bovine albumin fatty acid-free and the fatty acids (1) palmitic acid (C16:0), (2) stearic acid (C18:0), (3) oleic acid (C18:1), (4) arachidic acid (20:0), (5) arachidonic acid (20:4), (6) eicosapentaenoic acid (20:5), (7) dosapentanoic acid (C22:5). (8) docosahexaenoic acid (C22:6), (9) lignoceric acid (C24:0) nervonic acid (C24:1), and (10) heptadecanoic acid (C17:0), served as an internal standard, and were purchased from Sigma Aldrich.
The following antibodies were acquired from Beckman Coulter CD3-CD16/CD56+, anti- CD158a anti-KIR2DL1/S1 (clone EB6B and clone Z27.3.7), anti-CD94 (clone HP-3B1) and anti NKG2A (CD159a, clone Z199). From Becton Dickinson, were purchased following antibodies: anti-KIR3DL1 (clone DX9), anti NKp30 (clone P30-15), anti NKp46 (clone 9E2), anti CD160 (clone BY55). Anti-human anti-CD158b (anti-KIR2DL3, clone DX27), CD 158f (anti-KI2DL5, clone UP-R1), anti-NKP44 (clone P44-8), and anti CD161 (HP-3G10) were purchased from Biolegend. The anti NKG2D (Clone # 149810) was purchased from R&D Systems. The antibodies anti-CD158e1/e2 KIR3DL1 (clone REA168), anti-KIR2DS4/CD158f (clone JJC11.6) were purchased from Miltenyi Biotech. The anti-mouse Fab FITC, PE, PE-Cy7 and the isotype controls mouse IgG1, IgG2 labelled with FITC, PE or PC5 were purchased from Becton Dickinson.

The cell proliferation was measured using the EZ-Cytox Cell viability assay kit (Daeillab Service, Seoul, Korea). For blocking of NKG2D and 2B4, NK cells were preincubated with or without 5 μg/ml anti-NKG2D (clone 149810; R&D systems) and/or 5 μg/ml anti-2B4 (clone C1.7; BioLegend) for 30 min at 4 °C. After that, 100 μl of NK cells (10⁴ cells/well) was added to each well of a 96-well plate with or without feeder cells (10⁶ cells/well). After 5 days, 10 μl of the EZ-Cytox solution was added to each well of the plate and incubated at 37 °C for 4 h. The absorbance was measured by means of the SoftMax Pro 4.3.1 software (Molecular Devices, Sunnyvale, CA) at 450 nm.

The CD107a degranulation assay was performed as described previously (1 (link)). Briefly, 10⁵ cells of γδT-cell lines starved overnight of interleukin-2 (IL-2) or freshly collected PBMCs were incubated in the presence of stimulant and anti-CD107a-PE antibody (BD Bioscience) for 4 h. Cells were collected, washed, labeled with anti-Vδ2-FITC antibody (Beckman Coulter), and analyzed by flow cytometry. For transwell experiments, cell culture inserts (0.4-μm polycarbonate membrane; the Transwell system; Nunc, Roskilde, Denmark) were used by following the manufacturer's recommendations, with γδT cells seeded in the bottom of the wells in the presence of anti-CD107a-PE antibody and midstage schizonts (38 to 40 hpi) in the upper chamber. For antibody blocking experiments, γδT cells were preincubated for 1 h with anti-Vδ2 (clone immu389; Beckman Coulter), anti-BTN3A (clone 103.2; kind gift from D. Olive [21 (link)]), or anti-NKG2D (clone 149810; R&D Systems) antibody before stimulation as described previously (1 (link)).