Staining Neutrophils With Anti-Mouse GM-CSF R Alpha

September 25, 2018

Pediatrics
UW-Madison
Postdoc

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Company:

R&D Systems

Product Name:

Anti-mouse GM-CSF Receptor Alpha- PE

Catalog Number:

FAB6130P

I stained neutrophils for GM-CSF receptor alpha. These neutrophils were differentiated from murine granulocyte-macrophage progenitors. Previously, these neutrophils have been shown to be receptive to GM-CSF in culture. In planning to mutate GM-CSF receptor in the progenitor cells, which are passaged in vitro, I stained progenitors and differentiated neutrophils with anti-GM-CSF Ra-PE. I stained cells with two separate protocols to stain just surface receptors and after permeabilizing cells to stain all GM-CSF Ra. I show just the data from the neutrophils, the progenitors did not stain positive for GM-CSF Ra by either protocol.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Neutrophils

Primary Incubation

For surface stain, titrations of anti-GM-CSFRa completed as shown. Surface staining for both protocols contained antibodies to gate for neutrophils.

Blocking Agent

Buffer contained 0.5% BSA and FcBlock.

Secondary Incubation

For intracellular staining, cells were fixed with BD Fix/Perm buffer.

Tertiary Incubation

For intracellular staining, anti-GM-CSFRa-PE was incubated for 30 minutes at room temperature in BD Perm/Wash buffer.

Detection

Fluorescence by flow cytometry.

Results Summary

Surface staining of neutrophils with anti-GM-CSFRa showed no positive staining irrespective of the amount of antibody used for staining. Intracellular staining was successful at staining neutrophils. Unfortunately, the histograms of unstained and stained cells greatly overlap making it next to impossible to clearly distinguish cells that are positive or negative for GM-CSFRa.

DOI or PMID #

N/A

Additional Notes

In GM-CSF receptor deficient neutrophils, this antibody does not stain neutrophils (using intracellular staining protocol)

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Summary

The Good

Does stain GM-CSF receptor alpha

The Bad

Doesn't stain well, and, at least for neutrophils, requires intracellular staining

The Bottom Line

There aren't many options for staining murine GM-CSF Ra. Cytokine receptors are also tricky to stain well. While the uses of this antibody are limited, it does appear to be specific and works for basic applications.

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