CD25-169Tm Works Great With Proper Titration/Panel Optimization

Rheumatology
Vanderbilt University
Postdoctoral Fellow

Overall

Quality of Results

Ease-of-Optimization

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Company:

Fluidigm

Product Name:

Anti-Human CD25-169Tm

Catalog Number:

3169003B

 

Browse Similar Products: CD25 Flow Cytometry Antibodies

Our group studies T cells in the blood of untreated chronic HIV+ individuals. We are constantly expanding and optimizing our T cell panels to measure various T cell subsets, activation, and inhibitory receptor phenotype. We recently have begun optimizing mass cytometry panels to expand our panels significantly. We chose CD25-169Tm because it was commercially available.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human PBMC

Primary Incubation

0.25uL CD25-169Tm in 100uL PBS with 10^6 cells; 25 minutes; room temperature

Blocking Agent

None.

Secondary Incubation

None.

Tertiary Incubation

None.

Detection

CyTOF (Fluidigm)

Results Summary

The attached image illustrates the importance of having fluorescence cytometry controls to compare to when titrating antibodies for mass cytometry. Antibodies should be titrated individually for optimal stain index by fluorescence cytometry. Then, cells from the same individual should then be stained in tandem with fluorescently-conjugated CD25 and various volumes of CD25-169 to determine which volume of CD25-169 to pick. As illustrated in the image, increasing volumes of CD25-169 can quickly increase false positive signal that is less obvious than it is in fluorescence cytometry. At 0.25uL, CD25-169Tm works great to detect CD25+ cells from human PBMC samples after overnight SEB stimulation (1ug/mL).

Additional Notes

None.

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Summary

The Good

0.25uL/sample of CD25-169Tm works great!

The Bad

Nothing wrong with product; just be sure to titrate properly!

The Bottom Line

CD25-169 works great and is a nice option for incorporating CD25 into a mass cytometry panel.

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