Flow Cytometric Detection of Apoptosis (Chromatin Condensation)

February 16, 2016

LMU Munich
Anaesthesiology
Researcher

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Company:

Thermo Fisher Scientific

Product Name:

Violet Chromatin Condensation/Dead Cell Apoptosis Kit with Vybran DyeCycle Violet and SYTOX AADvanced for Flow Cytometry

Catalog Number:

A35135

In this review we evaluate an easy-to-use flow-cytometry-based detection method for apotosis. We use this for a first screening of cell-culture cells.

Experimental Design and Results Summary

Application

Flow Cytometry

Starting Material

Cultured cells or primary PBMCs

Protocol Overview

Induce apoptosis using the desired method and incubate the desired time. Stain 1 million cells in 1ml with SYTOX AADvanced (Stock: 500µM, 1µl per ml) to exclude dead cells. Stain with Dye Cycle Violet Stain (1µl per ml). Incubate for 30 minutes on ice in the dark. Detect on flow cytometer using violet and blue laser without washing.

Tips

Use HBSS for cell suspension, incubate on ice in the dark. Do not wash after incubation.

Results Summary

Apoptosis can be detected by fluorescence shift. This highly depends on the cells used. In cultured cells (U87 Glioblastoma, see Picture attached) this works really well. Using PBMCs, the shift of fluorescence is substantially smaller.

Additional Notes

Store at 2-6°C, do not freeze!

Image Gallery

Summary

The Good

Quick screening method, fast (therefore suitable for sensitive cells), dead cell exclusion possible.

The Bad

Only 1 property of apotosis is assayed by this method (chromatin condensation)

The Bottom Line

Quick screening method for Chromatin condensation including dead cell exclusion. Cost effective (200 assays for approx. 299 $). Other antibodies can be co-stained.

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