Live dead discriminator

dNK cells were gated on as live, CD9⁺CD56⁺ cells. pbNK cells were gated on as live CD56⁺CD3⁻ cells. The following Abs were used: Live/Dead discriminator (Life Technologies), CD9 (SN4 or M-L13 from eBioscience or BD Biosciences, respectively), CD56 (HCD56 from BioLegend), and CD3 (SK7) from BD Biosciences. Fibroblasts and macrophages were identified using CD10 (HI10a from BioLegend) and CD14 Abs (MφP9 and HCD14 from BD Pharmingen and BioLegend), respectively. The following Abs were used to stain KIRs: UPR1 (KIR2DL5) from BioLegend and Carlos Vilches (33 (link)); 179315 (KIR2DS4), 143211 (KIR2DL1), and 181703 (KIR2DL4) from R&D Systems; FES172 (KIR2DS4) and EB6 (KIR2DL1/S1) from Beckman Coulter; CHL (KIR2DL2/3/S2) from BD Pharmingen; DX9 (KIR3DL1) from BioLegend; NKVFS1 (KIR2DL1/2/3/S1/2/4) from Abcam; 5.133 (KIR3DL2) from Marco Colonna (34 (link)); and FLAG Abs from Sigma-Aldrich. Intracellular staining was performed according to the manufacturers’ instructions with Abs against Ki647 (BD Pharmingen), CCL3 (R&D Systems), and GM-CSF (BD Biosciences).

For baseline quantification of surface receptors, blood leukocytes were stained and analyzed using flow cytometry as previously described [16 (link)]. Whole blood samples were collected into K₂EDTA vacutainers (BD Biosciences) during the same blood draw described above. Samples were centrifuged at 400x g for 10 min at 4°C, plasma was removed, and cells were resuspended to the original volume in ice cold PBS containing 2.5 mM EDTA. Cells were kept on ice and in the dark for the remaining steps. Cells were incubated with a live/dead discriminator (Life Technologies) and an Fc-blocking reagent (TruStain FcX, BioLegend) for 15 min before an antibody cocktail was added for an additional 15 min. The antibody cocktail consisted of antibodies against CD16 (clone 3G8, BioLegend), CD14 (clone 61D3, eBioscience), CXCR2 (clone 5E8-C7-F10, eBioscience), activated CD11b (clone CBRM1/5, eBioscience), CD63 (clone H5C6, eBioscience), and CD66b (clone G10F5, eBioscience). Live, singlet cells were analyzed using a LSR Fortessa flow cytometer (BD Biosciences) and gated based on forward scatter, side scatter, and surface marker expression (neutrophils are defined as CD16^High FSC^High SSC^Mid, monocytes as CD14^High CD63^High FSC^Mid SSC^Mid, and NK cells as CD16^Mid FSC^Low SSC^Low).