Proliferation Assay for Flow Cytometry Using Edu

October 12, 2015

LMU Munich
Anaesthesiology
Researcher

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
Write a Review

Company:

Thermo Fisher / Life Technologies

Product Name:

Click-iT® EdU Pacific Blue™ Flow Cytometry Assay Kit

Catalog Number:

C-10418

We set out to investigate the proliferative properties of U87 Glioblastoma cell lines. Since we were not convinced using BrdU Assays because of harsh conditions to get BrdU accessible for antibody binding (such as heat, DNAse, etc), we chose the EdU assay on a flow cytometry platform. We also found that BrdU signals seem to alter during these harsh treatments. In addition, we did not want radioactivity in our lab, so we refrained from using a radioactive labeled Thymidine analog. The EdU procedure was characterized well and easy to establish in our lab. Therefore we chose the elegant approach of cell labeling with EdU and staining the EdU-labeled cells with a fluorescent dye binding to EdU (in this case Pacific Blue).

Experimental Design and Results Summary

Application

Flow Cytometry

Starting Material

U87 Glioblastoma cell line

Protocol Overview

Prior to harvesting, add EdU according to manufacturer's instructions and incubate for 2 hours. Harvest cells and wash with PBS. Permeabilize the cells for accessing the EdU with triton X-100 for 30 minutes. Saponin is also supplied with the kit. Wash with PBS 1%BSA. Add antibody in reaction buffer and incubate in the dark for 30 minutes. Wash with PBS 1%BSA twice. Analyze on a flow cytometer.

Tips

After fixation, the cell pellet is very loose, be careful when pipetting away the supernatant.

Results Summary

After labeling with EdU and staining EdU labeled cells with the Pacific Blue antibody, you can see clearly the EdU-positive (S-Phase) cells.

Additional Notes

Cell size and granularity is affected by permeabilizing. Perhaps gating adjustment is needed.

Related Categories

Image Gallery

Summary

The Good

Easy to use, reliable, not many cells needed.

The Bad

Expensive, Fix and perm times, washing steps.

The Bottom Line

This is a very reliable tool with exact and reproducible readouts, especially for a quick screening of the bottom readout regarding proliferation. Because they are exact and reproducible, results are also suitable for the detection of small differences.

Join the discussion