CD32A-Specific Antibody For Flow Cytometry

March 08, 2017

Boston Children's Hospital

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Company:

Stem Cell Technologies

Product Name:

Anti-Human CD32A Antibody, Clone IV.3, FITC

Catalog Number:

60012FI

Fc receptors mediate cellular responsiveness to antibodies. Crosslinking of activating Fc receptors, such as CD32A, by IgG antibodies bound to antigen can trigger anaphylaxis. We sought to assess Fc receptor expression on human peripheral blood mononuclear cells. In people, CD32 (FcgRII) exists as three genes: CD32A, CD32B and CD32C. A and C are activating while B is inhibitory. Differentiating these proteins is difficult due to the high level of conservation in the extracellular domains. This clone, IV.3, recognizes an extracellular epitope on CD32A and CD32C but not CD32B, making it very useful for flow cytometry. At the moment, Stem Cell Technologies appears to be the only supplier of a fluorescently conjugated form of this antibody, and it is only available in FITC.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human peripheral blood

Primary Incubation

20ul of antibody for 100ul whole blood for 30min at room temperature

Blocking Agent

5ul FcX Trustain (Biolegend, 422302)

Secondary Incubation

None

Tertiary Incubation

None

Detection

Flow Cytometry: BD LSR Fortessa, FITC channel

Results Summary

Staining appears in a pattern that is consistent with specificity for CD32A/C and not CD32B. Adequate staining on monocytes (CD3-CD19-) and basophils (CCR3+FceRI+), both of which express CD32A by qPCR. Minimal background on CD3+ T cells or CD19+ B cells, which do not. FITC conjugate is obviously not very bright. Lower amounts of antibody reduced the staining intensity, while higher amounts started to shift the background on all cells.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Specific, only readily available reagent for flow

The Bad

FITC conjugate is not bright, antibody is supplied in a relatively dilute solution

The Bottom Line

Adequate, specific, easy to use. Could easily be improved by conjugation with a different fluorophore.

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