Flow cytometric analysis of live human peripheral blood mononuclear cells using CD16 (3G8) Mouse mAb (PE Conjugate) and co-stained with NCAM1 (CD56) (MY31) Mouse mAb (APC Conjugate) #51997 (right), compared to concentration-matched Mouse (MOPC-21) mAb IgG1 Isotype Control (PE Conjugate) #63630 (left).
|Isotype||Mouse IgG1, kappa|
This Cell Signaling Technology antibody is conjugated to PE and tested in-house for direct flow cytometric analysis in human cells.
Supplied in 10 mM NaH2PO4, 150 mM NaCl, 0.09% NaN3, 0.1% gelatin, pH 7.2. Store at 4ºC. Do not aliquot the antibody. Protect from light. Do not freeze.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.
NOTE: Count cells using a hemocytometer or alternative method.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to Immunostaining.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
posted June 2017
revised August 2019
Protocol Id: 1504
CD16 (3G8) Mouse mAb (PE Conjugate) recognizes endogenous levels of total CD16 protein. This antibody detects an epitope within the extracellular domain.Species Reactivity:
This monoclonal antibody was purified from tissue culture supernatant via affinity chromatography. The purified antibody was conjugated under optimal conditions, with unreacted dye removed from the preparation.
CD64 (FcgammaRI), CD32 (FcgammaRII) and CD16 (FcgammaRIII) are three classes of the immunoglobulin superfamily. CD64 has a high affinity for IgG with three Ig-like domains while CD32 and CD16 have low affinities with two Ig-like domains. Two genes encode CD16-A and CD16-B resulting only in a 6 amino acid difference in their ectodomains. However, CD16-A has a transmembrane anchor versus CD16-B, which has a glycosylphosphatidylinositol (1). CD64, CD32 and CD16 are membrane glycoproteins that are expressed by all immunologically active cells and trigger various immune functions (activate B cells, phagocytosis, antibody-dependent cellular cytotoxicity, immune complex clearance and enhancement of antigen presentation) (2). CD16 cross-linking induces tyrosine phosphorylation (Tyr394) of Lck in NK cells (3). CD32 has tyrosine-based activation motifs in the cytoplasmic domain in contrast to CD16, which associates with molecules possessing these motifs (1).
CD16A is expressed by NK cells, macrophages, and a subset of monocytes, while CD16B is expressed by neutrophils (4). CD16 is commonly used in combination with CD56 to characterize NK cells, with CD16 identifying NK cells capable of cytotoxicity (5).
The 3G8 antibody is widely used as a marker of CD16 expression on the cell types mentioned above (6).
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