Low-Expressing Cells Difficult To Quantitate

September 27, 2018

Immunology
Columbia University
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

Novus Biologicals

Product Name:

Adenosine A2a R Antibody (7F6-G5-A2) [PE]

Catalog Number:

NBP1-39474PE

As a part of a preliminary experiment, we wanted to characterize the surface expression of A2AR on the human prostate line DU145. Looking at the data available regarding mRNA expression of the ADORA2A (A2AR) gene, DU145 is shown to be on the lower side of expression, so we wanted an antibody that would be bright enough to distinguish expression on the surface - hence choosing an antibody fluorescing in PE. The staining seemed to peak at around 2.5 uL/test in this cell line - going higher did not result in proportionate changes to the staining intensity.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

DU145 Cell Line

Primary Incubation

5 uL/test (50 uL) at RT for 20 mins

Blocking Agent

None

Secondary Incubation

None

Tertiary Incubation

None

Detection

Flow Cytometry

Results Summary

DU145 cells were stained with this A2AR antibody at 5uL/test in a volume of 50 uL. They were incubated in the stain for 20 minutes (stain also contained viability dye in APC Cy-7), and then washed and run on the flow cytometer. Analysis was done on only live cells. At the concentration of 5uL/test, you can see that there is a slight shift in the peak versus the unstained population. Going further up in terms of concentration (10uL/test) did not shift the peak much further, and 2.5uL/test did not result in much of a decrease in the peak intensity. However, using anything below that (1uL and below) caused the peaks to be indistinguishable from the non-stained sample.

DOI or PMID #

N/A

Additional Notes

A2AR can be inducible in some cell lines to increase expression.

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Summary

The Good

Can stain cells with low expression of A2AR

The Bad

Takes a lot of antibody per test

The Bottom Line

This is a good antibody for detecting even low levels of A2AR expression, but it is not really sustainable for use in large assays due to the cost and the amount required for each test. The staining is clear and is easy to compensate. For low expressing cell lines, it takes a bit of effort and time to titrate this antibody. Will probably be trying other companies' A2AR antibodies in other colors to see if any are brighter at high concentrations.

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