EdU incorporation for DNA synthesis and cell cycle analysis

January 16, 2015

Boston University
Pharmacology
Senior Post-doc

Overall

Quality of Results

Ease-of-Optimization

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Company:

Life Technologies

Product Name:

Click-iT® Plus EdU Flow Cytometry Assay Kits, 100 tests

Catalog Number:

C10635

Traditional BrdU labeling for DNA synthesis and S phase analysis requires DNA denaturation to expose BrdU for antibody detection. This kit uses Clik-it reaction to detect the incorporated EdU, an alternative of BrdU, without denaturing DNA and using antibody. Very useful for direct detection of DNA synthesis and assessing cell cycle progression.

Experimental Design and Results Summary

Application

Flow Cytometry

Starting Material

U2OS cell line

Protocol Overview

Basically follow the protocol downloaded from the company's web site. The protocol is very detailed and easy to follow. There are a few key points for using this kit: 1. Labeling with 10uM EdU for just 30 minutes is sufficient. 2. Fixation is critical to obtain good result. I use 75% EtOH to fix instead of using the fixative agent that comes with this kit. 3. Permeabilization is important for Click-it reaction.

Tips

None

Results Summary

The review picture showed the dual parameter plots of unlabeled and labeled samples. Cells labeled with EdU showed the proliferative cells in S-phase of cell cycle (green arch in the plot).

Additional Notes

None

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Summary

The Good

Easy, convenient, and sensitive

The Bad

A bit pricey.

The Bottom Line

This kit is very sensitive and useful for analyzing the DNA synthesis and cell cycle progression. Skipping the traditional DNA denaturation and antibody probing is the best part of using this kit.

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