Anti-CD73 with Strong Staining and Good Clarity

September 05, 2018

BlueskyReddit
Immunology
Columbia University
Graduate Student

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Company:

BioLegend

Product Name:

APC anti-human CD73

Catalog Number:

344005

Human cell lines express varying amounts of CD73, and this antibody stains very brightly. It took a little bit of work to titrate and find the optimal concentration, and it seems like the antibody has to be re-titrated for every cell line. For the human line DU 145, I started at a 1:50 titration and went down to 1:1000 - I found that 1:700 works very well for this particular line, which is very dilute for an anti- human anitbody.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

DU145 Cell Line

Primary Incubation

1:700 at RT for 20 mins

Blocking Agent

None

Secondary Incubation

None

Tertiary Incubation

None

Detection

Flow Cytometry

Results Summary

For this experiment, we compared the expression of CD73 on DU145 cell lines to the FMO in order to determine whether the cells expressed CD73 and what the expression looked like. The cells were stained with Live/Dead in addition to CD73, and results were gated on live cells only. CD73 in APC was used at 1:700. There is a significant shift in the fluorescence of APC-CD73 on the cell line compared to the FMO, indicating that there is indeed expression of CD73 on the surface of these cells. Additionally, the cells all seem to express CD73 uniformly at rest.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Very strong stain

The Bad

Needs to be re-titrated for each cell line according to baseline levels

The Bottom Line

This is a very strong antibody which can be used for quite some time due to how dilute it can be used. I titrated it out in DU145 at 1:700, after using it at 1:25 and 1:50 as is advised with most human antibodies and finding that the peak was way off scale. However, other cell lines, including LnCap, seem to express less CD73 and more antibody has to be used to gauge shifts in the expression. It's easy to comp due to its strong and even staining.

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