To quantify various NK cell populations in the PBMCs, 5 × 105 cells were stained with various combinations of fluorophore-conjugated monoclonal antibodies (mAbs). The Gating process of CD3−CD16brCD56dimNK cells was shown in Figure S1 , according to the previous report (36 (link)). Stained cells were fixed with 4% paraformaldehyde and examined by FACSCalibur (BD Biosciences), and the data analyzed Cell Quest Pro software (FlowJo, LLC, Ashland, OR). A total of 50,000 lymphoid events were acquired in each sample. Cells were phenotypically analyzed by staining for 20 min with the following fluorophore-conjugated anti-human mAbs: fluorescein isothiocyanate (FITC)-αCD3, PerCP eFluor710-αCD16, phycoerythrin (PE)-CD56, eFlour660-CD107a, APC-CD158 (KIR2D L1/S1/S3/S5), APC-CD158e1 (KIR3DL1), APC-CD159a (NKG2A), APC-CD226 (DNAM-1), APC-CD244 (2B4), APC-CD314(NKG2D), AF647-CD337 (NKp30), and APC-anti-HLA-E (all from BioLegend, eBioscience, and Miltenyi Biotec). Positive staining populations were determined by comparison with isotype controls. To evaluate HLA expression on CD45+CD34+ myeloid progenitor cells, the cells were stained with PerCP Cy5.5-CD45 (BioLegends), PE-CD34 mAbs (BD Biosciences), and APC-anti-HLA-E mAb, and examined by flow cytometry.
Apc cd314
Manufactured by BioLegend
Sourced in United States
APC-CD314 is a fluorescence-labelled monoclonal antibody that binds to the CD314 antigen, also known as NKG2D. CD314 is a type II transmembrane protein that is expressed on the surface of natural killer (NK) cells, CD8+ T cells, and other immune cell types. The APC fluorophore is conjugated to the antibody, providing a detectable signal for flow cytometry analysis.
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2 protocols using apc cd314
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Quantification of NK Cell Populations in PBMCs
2
Quantification of circulating NK cells
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The following antibodies were used to stain peripheral blood mononuclear cell (PBMC) suspensions: fluorescein isothiocyanate (FITC)-CD3, FITC-CD4, peridinin-chlorophyll-protein (PerCP/cyanine 5.5-CD16, phycoerythrin (PE)-CD19, PE-CD56, adenomatous polyposis coli (APC)-CD158 (KIR2D L1/S1/S3/S5), APC-CD158e1 (KIR3DL1), APC-CD159a (NKG2A), APC-CD226 (DNAM-1), APC-CD244 (2B4), APC-CD314 (NKG2D), and Alexa Fluor (AF)647-CD337 (NKp30) (BioLegend, San Diego, CA, USA), eFluor660-CD107a (eBioscience, San Diego, CA, USA), and CD8 (BD Biosciences, Franklin Lakes, NJ, USA). The subpopulation of circulating NK cells was quantified and analyzed using flow cytometry, as described previously.32 (link)
Briefly, 5 × 105 PBMCs after red blood cell lysis were collected for incubation with antibodies for 30 minutes at 4°C, washed with phosphate-buffered saline and fixed with 4% paraformaldehyde for 20 minutes. The cells were then washed twice with phosphate-buffered saline and analyzed using FACSCalibur (BD Biosciences). Data were analyzed using Cell Quest Pro (FlowJo, LLC, Ashland, OR, USA). Human plasma IL-6 and TNF-α levels were measured with commercially available Quantikine Enzyme-Linked Immunosorbent Assay Kits (R&D Systems, Inc., Minneapolis, MN, USA) in accordance with the manufacturer’s instructions.
Briefly, 5 × 105 PBMCs after red blood cell lysis were collected for incubation with antibodies for 30 minutes at 4°C, washed with phosphate-buffered saline and fixed with 4% paraformaldehyde for 20 minutes. The cells were then washed twice with phosphate-buffered saline and analyzed using FACSCalibur (BD Biosciences). Data were analyzed using Cell Quest Pro (FlowJo, LLC, Ashland, OR, USA). Human plasma IL-6 and TNF-α levels were measured with commercially available Quantikine Enzyme-Linked Immunosorbent Assay Kits (R&D Systems, Inc., Minneapolis, MN, USA) in accordance with the manufacturer’s instructions.
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