Good Option For MHC Class II Staining In A Full Cocktail

September 21, 2016

Pediatrics
UW-Madison
Postdoc

Overall

Quality of Results

Ease-of-Optimization

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Company:

BioLegend

Product Name:

APC/Fire-750 anti-Mouse I-A/I-E

Catalog Number:

107652

When staining for DCs (and other MHC class II-expressing cells) in inflamed mouse lung using a full cocktail to track many different markers, the high expression of MHC class II on mature DCs is very high often pushing the events to the far end of the flow plot/histogram. To avoid this turning the voltage down on the channel can work, but becomes very difficult with the more fluors used when attempting to complete compensation. The APC/Fire750-conjugated antibody recognizing murine MHC class II (I-A/I-E) is much more stable than APC-Cy7 but is dimer than most fluors making it very nice for looking at cells with high MHC class II surface expression.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Lung digests of inflamed mouse lung

Primary Incubation

1/200 dilution for at least 20 minutes at 4C

Blocking Agent

Buffer contained BSA and FcBlock

Secondary Incubation

None

Tertiary Incubation

None

Detection

Fluoresecence in flow cytometry

Results Summary

This antibody clone works well for detecting MHC class II on murine leukocytes regardless of conjugated fluor. Figure shows two different stains of murine pulmonary leukocytes stained with I-A/I-E conjugated to either APC/Fire-750 or Alexa647. Population shown is Ly6G-, Siglec F-, and CD11b+ so is mostly monocytes (CD11c-, MHCII-) and DCs (CD11c+, MHCII+). For these two stains, there is no major difference in using either fluor conjugate, but in more complex flow panels, the APC/Fire750 has been very useful for this particular antibody.

Additional Notes

Not worth the extra cost for a simple flow experiment (when tracking fewer markers)

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Summary

The Good

Antibody clone is very good, fluor conjugate is stable, useful in a packed staining cocktail

The Bad

More expensive than many other fluor conjugates of this antibody

The Bottom Line

This reagent is great if you are tracking many populations or tracking many features of antigen presenting cells. If you just need this antibody for a simple cocktail, then FITC or other less expensive conjugates will work fine.

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