Pac Blue Anti-Siglec H staining for pDCs

April 12, 2017

Pediatrics
UW-Madison
Postdoc

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Company:

BioLegend

Product Name:

Pacific Blue™ anti-mouse Siglec H Antibody

Catalog Number:

129609

Plasmacytoidal dendritic cells (pDCs) are characterized in mice as expressing CD11c (on most DCs) and the B cell marker B220. A unique marker to identify pDCs in mice is Siglec H. In this experiment I titrated anti-Siglec H Pac Blue on splenocytes from a naive C57B/6 mouse to determine the optimal staining concentration of anti-Siglec H for pDCs.Biolegend indicates that a 1:200 dilution is optimal for this product but shows a small separation of Siglec H positive and negative populations on their site's data (also from splenocytes). To improve visualization of the data, I have pregated the population based on other markers expressed by pDCs (CD11c+, Ly6C+, CD11b-, F4/80-). I saw that 1:200 dilution of anti-Siglec H was enough to see a positive (pDC) population, but there was better separation of peaks at 1:100 and 1:50 dilutions (optimal concentration appears to be 1:100).

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Splenocytes from naive C57B/6 mice

Primary Incubation

Titrations (as shown) for 30 minutes at 4C along with antibodies against other markers for gating.

Blocking Agent

Buffer contained BSA and FcBlock

Secondary Incubation

None

Tertiary Incubation

None

Detection

Fluoresecence in flow cytometry

Results Summary

Because anti-Siglec H is useful to indicate pDCs, I pre-gated a population before gating on B220 and Siglec H (CD11c+, Ly6C+, CD11b-, F4/80-). The antibody successfully stained the pDCs at all tested concentrations (1:50 to 1:200) but showed better separation of positive and negative populations at the higher concentrations. To save on antibody and limit non-specific staining, 1:100 dilution probably would work best.

DOI or PMID #

N/A

Additional Notes

Siglec H does not appear to be very highly expressed on the surface of pDCs, I would suggest using brighter fluorophores if applicable (Pac Blue worked well but PE, for example might work better)

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Summary

The Good

Anti-Siglec H-Pac Blue worked well for staining pDCs, reagent is reasonably priced

The Bad

Siglec H is not very highly expressed on the surface of pDCs, and it may not be necessary to stain for Siglec H to identify pDCs.

The Bottom Line

If you want to include Siglec H in your panel, this product will work for you.

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