Staining Murine Leukocytes With Anti-TLR4 Clone SA15-21

August 10, 2017

Pediatrics
UW-Madison
Postdoc

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Company:

BioLegend

Product Name:

APC-anti-mouse TLR4 (clone SA15-21)

Catalog Number:

145405

There are two main commercial clones that stain for TLR4 (CD284). MTS510 recognizes the TLR-4/M2 complex (together these bind LPS) while SA15-21 recognizes TLR-4 individually. I was interested in just TLR-4 expression on cells, so I tried this SA15-21 to surface stain cells. I first titrated the antibody on splenocytes from naïve mice gating on B cells in the concentration range suggested by BioLegend. I later stained DCs from lungs of mice challenged with a pulmonary fungal infection. APC-anti-mouse TLR4 (clone SA15-21) does detect TLR4 on the surface of cells, but the positive signal isn’t much brighter than the negative signal.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Murine splenocytes or lung leukocytes

Primary Incubation

In stain cocktail at concentrations indicated. 4C for 30 minutes in FACS buffer.

Blocking Agent

Buffer contained BSA and FcBlock

Secondary Incubation

Cells were fixed with 2% paraformaldehyde

Tertiary Incubation

None

Detection

Fluoresecence in flow cytometry

Results Summary

When I titrated APC-anti-mouse TLR-4 (clone SA15-21) on B cell splenocytes from naïve mice, I did not see a high proportion of cells that had stained positive with SA15-21, but a 1:100 dilution appeared to be the best concentration for staining. I used this concentration (1:100) to stain lung leukocytes from mice with a pulmonary blastomycosis infection. Looking at dendritic cells, I saw that DCs did have increased APC fluorescence compared to the isotype control when staining with APC-anti-TLR-4, but the shift in fluorescence was minor. This antibody can track TLR-4 expression, but a FMO-control is necessary to distinguish from the background fluorescence.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Does stain cells

The Bad

Positive staining not much brighter than negative; need an FMO control.

The Bottom Line

TLR-4 can be detected on the surface with APC-anti-mouse TLR-4 (clone SA15-21) but staining is not very bright. I would suggest using clone MTS510 if applicable.

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