Cd16 32 clone 93

Manufactured by BioLegend

The CD16/32 (clone 93) is a monoclonal antibody that binds to the murine Fc gamma receptors III and II (CD16 and CD32). It is commonly used in flow cytometry to block non-specific binding of antibodies to Fc receptors on mouse cells.

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5 protocols using cd16 32 clone 93

Comprehensive Murine Immune Cell Profiling

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Antibodies targeting the following murine proteins were purchased from BioLegend (San Diego, CA): PD-L1 (clone 10F.9G2), I-A/I-E (M5/114.15.2), CD45 (30-F11), CD19 (6D5), CD11b (M1/70), PD-1 (29F.1A12), TCRβ chain (H57-597), CD8a (53-6.7), PD-L2 (TY25), CD80 (16-10A1), CD4 (GK1.5), Ly6G (1A8), Ly6C (HK1.4), T-bet (4B10), ICOS (C398.4A), OX40 (OX-86), TCRγ/δ (GL3) and CD16/32 (clone 93). Antibodies targeting the following murine proteins were purchased from BD Biosciences (Franklin Lake, NJ): Siglec F (E50-2440), CD24 (M1/69), and CD3ε (145-2C11). Antibodies targeting the following murine proteins were purchased from eBioscience (Asheville, NC): iNOS (CXNFT), FoxP3 (FJK-16s), Ki67 (SolA15), CD11c (N418), Gata3 (TWAJ), and RORγt (B2D). A polyclonal antibody targeting murine arginase was purchased from R&D Systems. Isotype control antibodies were used according to manufacturer’s instructions in all experiments. Antibodies were conjugated to the following fluorophores: Allophycocyanin (APC), Alexa Fluor 700, APC-cyanine 7 (APC-Cy7), fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin-chlorophyll-protein (PerCP)-Cy5.5, PE-eFluor 610, PE-Cy5, PE-Cy7, Brilliant Violet (BV) 421, and BV 650.

Roussey J.A., Viglianti S.P., Teitz-Tennenbaum S., Olszewski M.A, & Osterholzer J.J. (2017). Anti-PD-1 Antibody Treatment Promotes Clearance of Persistent Cryptococcal Lung Infection in Mice. Journal of immunology (Baltimore, Md. : 1950), 199(10), 3535-3546.

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Cytokine Production in Mouse Splenocytes

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Mouse splenocytes were incubated with 5ng/ml mouse IL-2 (Biolegend), 1µl BD Golgiplug (Becton Dickinson, Franklin Lakes, NJ) and 10µg peptide (linear peptide sequences: PA D630: KGVED/GGSIG, PB1 E178: DKEE/KMEITT, NP 147: TYQRTRALV) in 96-well V-bottom plates at 37◦C, 5% CO₂ for 5 h. Cells were then stained with Zombie Aqua Fixable Viability kit (Biolegend) for 30 min, washed once and subject to antibody staining for 30 min with the following panel: purified CD16/32 (clone 93); CD3 PE/Dazzle 594 (clone 17A2); CD4 APC/Cy7 (clone GK1.5); CD8α PerCP/Cy5.5 (clone53-6.7); CD8β PerCP/Cy5.5 (clone YTS 156.7.7, all from Biolegend). Cells were washed once before fixation in 4% formaldehyde, and permeabilization with perm buffer (BD Bioscience), followed by intracellular staining with IFNγ FITC (clone XMG1.2, Biolegend). Samples were acquired by flow cytometry on BD LSRFortessa and analyzed using Flowjo. Total cytokine production was calculated by subtracting non-specific background using unstained controls.

Chu J.T., Gu H., Sun W., Fan R.L., Nicholls J.M., Valkenburg S.A, & Poon L.L. (2022). Heterosubtypic immune pressure accelerates emergence of influenza A virus escape phenotypes in mice. Virus Research, 323, 198991.

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Multiparameter Immune Cell Analysis

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Antibodies specific for murine CD183 (CXCR3, clone CXCR3 173), CXCL9 (clone MIG 2F5.5), IFNγ (clone XMG1.2), CD4 (clone RM4-5), CD8α (clone 5H10-1), CD45 (clone 30-F11), CD11c (clone N418), Ly6G (clone 1A8), CD335 ([NKp46], clone 29A1.4), CD90.1 (Thy1.1, clone OX-7), CD31 (clone 390), and CD16/32 (clone 93) were obtained from BioLegend. Anti-Adenosine A_2a receptor (ADORA2A) (clone 7F6-G5-A2) was obtained from Novus Biologicals.

Clancy-Thompson E., Perekslis T.J., Croteau W., Alexander M.P., Chabanet T.B., Turk M.J., Huang Y.H, & Mullins D.W. (2015). Melanoma induces, and adenosine suppresses, CXCR3-cognate chemokine production and T-cell infiltration of lungs bearing metastatic-like disease. Cancer immunology research, 3(8), 956-967.

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Murine IL-22 Immune Response Analysis

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Recombinant murine IL-22 was purchased from Peprotech (Cranbury, NJ, USA). Purified anti-mouse CD3ε (145-2C11) and its isotype control Armenian hamster IgG for the injection were obtained from BioLegend (San Diego, CA, USA) and BioXCell (Lebanon, NH, USA), respectively. Purified monoclonal anti-mouse IL-22 (IL22JOP) and its isotype control rat IgG2a used for in vivo administration were purchased from eBioscience and BioXCell, respectively. For immunohistochemistry, rabbit polyclonal anti-claudin-1 antibody was purchased from AbCam (Waltham, MA, USA). For flow cytometry, fluorescence conjugated antibodies specific for CD45 (clone 30-F11), CD4 (GK1.5), CD8α (536-7), CD19 (1D3), TCR-β (H57-597), IL-22 (Poly5164) and CD16/32 (clone 93) antibodies were purchased from BioLegend.

Zhou J., Onodera S., Hu Y, & Yu Q. (2022). Interleukin-22 Exerts Detrimental Effects on Salivary Gland Integrity and Function. International Journal of Molecular Sciences, 23(21), 12997.

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Evaluation of Immune Response Modulation

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Spleens were removed from vaccinated animals one week after the second boost, and splenocytes isolated aseptically using standard methods. 1 X 10⁶ splenocytes per well were stimulated in 96 well round bottom plates for 72 hours with 20 μg endotoxin-free recombinant SML-4 or SML-5 protein. Concanavalin A (MP Biomedicals) was added at a concentration of 1μg/mL as a positive control stimulus, and media alone served as a negative control. Supernatants were analyzed by Luminex (ProcartaPlex kits; ThermoFisher) according to the manufacturer’s instructions. After 72 hours, splenocytes were transferred to a fresh 96 well flat-bottomed plate containing bound α-CD3 (clone 17A2, Biolegend) and cells were restimulated for 6 hours along with soluble α-CD28 (clone 37.51, Biolegend) with the addition of Brefeldin A (Biolegend) in the last 4 hours of stimulation. After blocking with addition of 10 μg/mL CD16/32 (clone 93, Biolegend), cells were then surface stained for CD4 (PerCP-Cy5.5, clone GK1.5, Biolegend), CD44 (Alexa Fluor 700, clone IM7, eBioscience) and zombie live/dead (BV 510, Biolegend). After fixation and permeabilization using Fix/Perm buffer (BD) cells were intracellularly stained for IFN-γ (PE, clone XMG1.2, Biolegend) and IL-4 (Pe-Cy7, clone BVD6-24G2, eBioscience). Cells were read on an LSRFortessa X-20 flow cytometer (BD) and data analyzed using FlowJo software.

Srey M.T., Taccogna A., Oksov Y., Lustigman S., Tai P.Y., Acord J., Selkirk M.E., Lamb T.J, & Guiliano D.B. (2020). Vaccination with novel low-molecular weight proteins secreted from Trichinella spiralis inhibits establishment of infection. PLoS Neglected Tropical Diseases, 14(11), e0008842.

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