BrdU Flow Kit with Clear and Specific Staining

August 20, 2018

BlueskyReddit
La Jolla Institute for Allergy and Immunology
Developmental Immunology
Postdoctoral Fellow

Overall

Quality of Results

Ease-of-Optimization

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Company:

BD Biosciences

Product Name:

FITC BrdU Flow Kit

Catalog Number:

559619

The aim of the research was to measure proliferation of T cell subsets before and after bacteria infection. BrdU was injected IP at the time of infection, and mice were sacrificed 24 hours later. We followed the kit per instructions and obtained clear results.

Experimental Design and Results Summary

Application

BrdU labeling for flow cytometry

Starting Material

Collagenase-digested lung tissue

Protocol Overview

The mice were injected with BrdU 24 hours prior to sacrifice. Total cells were isolated, and stained with antibodies for surface markers. The cells were fixed and permed with BD cytofix/cytoperm. The cells were permeabilized with BD cytoperm, and then incubated a second time with cytofix/cytoperm. The cells were treated with DNAse, washed, and then stained with the FITC labeled BrdU antibody.

Tips

The second fix/perm step is very important. Do not fix the cells after intracellular staining. Not compatible with Ki-67 staining. Recommend a control without BrdU injected.

Results Summary

We obtained clear BrdU+ cells. The protocol did not affect any of our surface markers. We attempted to co-stain with Ki-67 antibody, but this antibody was not compatible with the BrdU protocol. It is possible other intracellular staining would require more optimization.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Excellent, straight forward, all inclusive kit for BrdU labeling of cells for FACS

The Bad

Relatively time consuming protocol, but there is an overnight stopping point if needed

The Bottom Line

This kit was great because it included everything I needed, and did not dampen the fluorescence of my cell surface antibodies as other protocols do. Co-staining with other intracellular markers may require further optimization.

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