Nkg2a clone 131411

Enumeration of NK/NKT-like cells and expression of activation and inhibitory receptors were investigated in samples from 56 acute and 31 convalescent patients and 56 control individuals. CD161 and inhibitory receptors were assessed only in acute patients and controls. Peripheral blood (100 μl) was stained according to a previously described protocol (Tripathy et al., 2011 (link)). The monoclonal antibodies used were anti-humanCD56 (clone B159), CD3 (clone SK7/UCHT1), NKp30/NKp44/NKp46/CD244/CD161/NKG2D (clone p30-15/p44-8/9E2/2-69/DX12/1D11), purchased from BD Biosciences and CD94 (clone 131412 APC), and NKG2A (clone 131411) purchased from R&D Systems Inc., MN, USA. NK (CD56⁺CD3⁻) and NKT-like (CD56⁺CD3⁺) cells were gated within the lymphocyte population on the basis of their CD56 and CD3 staining pattern; their receptors expression patterns are expressed as the percentage of gated NK and NKT-like cells.

Primary human NK cells were treated and stimulated as described and incubated for 5 days. Stimulated cells were washed and resuspended in flow buffer (0.5% fetal bovine serum/PBS) before cell surface staining was performed at 4°C, with fluorophore-conjugated antibodies against the following proteins: CD56 (clone HCD56, Biolegend), CD16 (clone 3G8, Biolegend), NKG2D (clone 1D11, BD Biosciences), CD57 (clone HNK-1, Biolegend), NKG2A (clone 131411, R&D Systems), CD94 (clone DX22, Biolegend), and DNAM1 (clone TX25, Biolegend). Isotype-matched control antibodies conjugated with the appropriate fluorophores were used in parallel (mouse IgG1, κ isotype control, clone MOPC-21, Biolegend; mouse IgM, κ isotype control, clone MM-30, Biolegend; mouse IgG1, κ isotype control, clone MOPC-21, BD Biosciences; mouse IgG2A isotype control, clone 20102, R&D Systems). Staining was performed with appropriate combination of fluorophores. Cell viability was assessed using a Live/Dead stain (Zombie NIR™ Fixable Viability kit, Biolegend). Cells were then washed and resuspended in flow buffer before fixation with 2% paraformaldehyde/0.25% fetal bovine serum/PBS at 4°C. Cells were analyzed by flow cytometry, and data analysis was performed using FlowJo_v10 software (Tree Star). Cell debris was excluded, and cells were gated on live single CD56⁺ pNK cells.