Nkg2a

Manufactured by Beckman Coulter

Sourced in Canada

The NKG2A is a laboratory equipment product manufactured by Beckman Coulter. It is a receptor protein that is expressed on the surface of natural killer (NK) cells and a subset of T cells. The NKG2A receptor plays a role in the regulation of the immune response.

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4 protocols using nkg2a

Multiparametric NK Cell Profiling

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Antibodies were purchased for CD16 biotin from BioLegend; LFA-1 open conformation isoform was from Abcam; pZAP/Syk, pS6, pSLP76, pAKT (S473), pPLCγ2, pERK1/2, and NF-κB pp65 were from BD; KIR2DL1/S1 was from Miltenyi; KIR2DL2/3/S2 was from Beckman Coulter; KIR3DL1/2 was from BioLegend; CD57 was from BioLegend; NKG2A was from Beckman Coulter; NKG2C was from Miltenyi; CD56 was from Beckman Coulter; CD16 was from BioLegend; 7AAD was from BD; dead-cell marker was from Life Technologies; and CD107a was from BioLegend. Pacific Orange and Blue Succinimidyl Ester were bought from Thermo Fisher Scientific. 5-Methylthioadenosine (MTA) and 3-deazaadenosine (3-Deaza) were purchased from Sigma-Aldrich, 5-azacytidine (5-Aza) was from Biomol/Cayman, and 2-chloroadenosine (CADO) and EPZ015666 (EZH) were from Sigma. Avidin was purchased from Sigma. K562 cell line from ATCC was cultured in RPMI 1640 media with antibiotics (penicillin/streptomycin; Invitrogen) and 10% heat-inactivated fetal calf serum (Sigma) at 37°C.

Jacobs B., Schlögl S., Strobl C.D., Völkl S., Stoll A., Mougiakakos D., Malmberg K.J., Mackensen A, & Aigner M. (2020). The Oncometabolite 5′-Deoxy-5′-Methylthioadenosine Blocks Multiple Signaling Pathways of NK Cell Activation. Frontiers in Immunology, 11, 2128.

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NK Cell Degranulation Assay

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Following overnight culture in 10³ U/mL IL-2, 5×10⁵ PBMCs were co-incubated for four hours with either 5×10⁴ parental 721.221 cells or 721.221 cells stably expressing HLA-B*44:03 in the presence of an APC H7 conjugated CD107a specific antibody (BD biosciences). To block inhibition imparted by the KIR3DL1, HLA-B*44:03 interaction, DX9 (20 µg/ml) was added to the co-incubation. Following this incubation, the cells were probed with conjugated antibodies specific for CD56 (Beckman Coulter), CD3 (Biolegend), KIR2DL2/L3 (BD Biosciences), KIR2DL1/S1 (Beckman Coulter), NKG2A (Beckman Coulter), ILT-2 (Beckman Coulter) and KIR3DL1 (Z27, Beckman Coulter). Using the differential surface expression phenotypes of the two KIR3DL1 alleles, the percent CD107a positive of each KIR3DL1 population was determined and adjusted for the percent CD107a positive of the KIR-/NKG2A- population to control for differences in the activation potential of the target cells. The data for each allele are expressed as a ratio comparing the degranulation responses observed against each target cell to the CD107a results observed following co-incubation with the HLA negative 721.221 cells.

Mulrooney T.J., Zhang A.C., Goldgur Y., Boudreau J.E, & Hsu K.C. (2015). KIR3DS1 specific D0 domain polymorphisms disrupt KIR3DL1 surface expression and HLA binding. Journal of immunology (Baltimore, Md. : 1950), 195(3), 1242-1250.

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Comprehensive Immunophenotyping of PBMCs

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For flow cytometry, PBMC samples were thawed in a 37°C water bath and resuspended in DMEM:F12 media (Fisher 11320–033). Cells were then pelleted (1000 rpm x 5 min) and washed twice using DPBS. Pellets were resuspended in live/dead solution (Fisher L34961) on ice for 20 minutes. Samples were then washed twice with FACS buffer and stained with extracellular antibodies consisting of CD14 (BD 561391), CD16 (BD 562874 and BD561394), CD11b (BD 561887), HLA-DR (BD 339194), CD3 (BD 558124 or BD 557757), CD20 (BD 560734), CD4 (BD 347327), CD8 (BD 335787), NKG2A (Beckman Coulter A60797), CD1c (Biolegend 331506), and CD123 (BD 560826) on ice for 30 min. Stained samples were washed twice with FACS buffer and fixed and permeabilized using BD Cytofix/Cytoperm (BD554714) for 20 min on ice. Samples were washed twice using 1x BD Cytoperm buffer (BD554714) 500 g x 4 min and resuspended in intracellular antibody cocktail consisting of CD38 (BD 560676) and Ki-67 (BD 558616) for 20 minutes on ice. After washing, samples were fixed and inactivated using 4% (w/v) paraformaldehyde, run on a BD LSRII, and analyzed using FlowJo 10.5.0.

Hartman A.L., Nambulli S., McMillen C.M., White A.G., Tilston-Lunel N.L., Albe J.R., Cottle E., Dunn M.D., Frye L.J., Gilliland T.H., Olsen E.L., O’Malley K.J., Schwarz M.M., Tomko J.A., Walker R.C., Xia M., Hartman M.S., Klein E., Scanga C.A., Flynn J.L., Klimstra W.B., McElroy A.K., Reed D.S, & Duprex W.P. (2020). SARS-CoV-2 infection of African green monkeys results in mild respiratory disease discernible by PET/CT imaging and shedding of infectious virus from both respiratory and gastrointestinal tracts. PLoS Pathogens, 16(9), e1008903.

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Comprehensive Immunophenotyping of NK Cells

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Thawed PBMC were stained with ethidium monoazide (EMA), anti-CD19-PeCy5, anti-CD3-AlexaFluor700 (both from BD Biosciences), and with either anti-CD14-V711 (Biolegend) or anti-CD14-PeCy5 (AbD Serotec, Raleigh, NC) to exclude dead cells, T cells, B cells and monocytes. NK cells were identified using anti-CD56-PeCy7 (BD Biosciences). FITC-conjugated antibodies against CD122, CXCR3 (R&D Systems), CD69 and HLA-DR (BD Biosciences), PE-conjugated antibodies against TRAIL, CD300 (BD Biosciences), NKG2A, NKG2D, and NKp44 (Beckman Coulter, Brea, CA), and APC-conjugated antibodies against CD85j (eBiosciences, San Diego, CA), CCR5 (BD Biosciences), NKG2C (R&D Systems), NKp30 and NKp46 (Miltenyi Biotec, Auburn, CA) were added. Liver-infiltrating lymphocytes were stained with anti-TRAIL-PE, anti-CD69-APC/Cy7 (BD Biosciences) and anti-NKp46-APC (Miltenyi Biotec) in addition to EMA and the lineage-specific antibodies described above.

Serti E., Chepa-Lotrea X., Kim Y.J., Keane M., Fryzek N., Liang T.J., Ghany M, & Rehermann B. (2015). Successful Interferon-Free Therapy of Chronic Hepatitis C Virus Infection Normalizes Natural Killer Cell Function. Gastroenterology, 149(1), 190-200.e2.

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