APC/Fire750 Works Well Has Many Applications For Neutrophils Conjugated To Anti-Ly6G

July 13, 2016

Pediatrics
UW-Madison
Postdoc

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Quality of Results

Ease-of-Optimization

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Company:

BioLegend

Product Name:

Anti-Ly6G-APC/Fire750TM

Catalog Number:

127652

Murine bone marrow neutrophils were enriched over a percoll gradient. To determine the purity of neutrophils, I stained with anti-CD11b and anti-Ly6G. APC/Fire750 is a new product and I wanted to see how it might work in my research, so I stained the same samples with anti-Ly6G conjugated to either PE or APC/Fire750 to see how well the new fluor conjugate works.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Murine enriched bone marrow neutrophils

Primary Incubation

1/200 dilution for 20 minutes at 4 C

Blocking Agent

0.5% BSA in staining buffer

Secondary Incubation

None

Tertiary Incubation

None

Detection

Fluorescence in flow cytometry

Results Summary

The anti-Ly6G-APC/Fire750 stained the neutrophils better than APC-Cy7 does (it appears to be more stable as promised by BioLegend). In terms of gating, the APC/Fire750 conjugate did as well as the PE conjugate to differentiate positive and negative populations. The APC/Fire750 is not very bright compared to most fluors but it is comparable to other fluors in its flow channel.

Additional Notes

While APC/Fire750 is not very bright, it may have many uses for anti-Ly6G. Ly6G is more highly expressed than most surface markers, so the decreased brightness might be beneficial. And added use for anti-Ly6G-APC/Fire750 is to use in tandem with a near-infrared viability dye to gate out neutrophils with dead cells in this difficult-to-use channel.

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Summary

The Good

Stable and stains well

The Bad

Fire makes the fluor sound bright, it really isn't that bright (but doesn't mean that it isn't useful)

The Bottom Line

I have many qualms about using APC-/Cy7, but I have this channel open for flow cytometry panels. The APC/Fire750 conjugate, because of its added stability, makes the high wavelength channel of the red laser more useful.

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