Informative Results, but Highly Cell Line–Dependent

December 03, 2018

BlueskyReddit
Princeton
Molecular Biology
Graduate Student

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Quality of Results

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Company:

STEMCELL Technologies

Product Name:

Aldefluor Kit

Catalog Number:

01700

Used this kit for quantifying the Aldefluor+ population in a number of different cell lines and primary cells. Two major observations were that (1) the recommended DEAB concentration is not sufficient to inhibit all ALDH isoforms, and that (2) 5 µL of the activated reagent per 1 mL buffer at 37°C for 40 minutes is ideal for most applications. Recommended times in the kit, particularly for cancer cells, are not sufficient.

Experimental Design and Results Summary

Application

Flow cytometry

Starting Material

Human and mouse breast cancer cells

Protocol Overview

Cells are trypsinized or isolated, then washed 2x with pbs. 5 µL of activated reagent is added to 1 mL of buffer. This is split and half is brought to 1 mM DEAB. This is added to the cells, incubated at 37°C for 40 minutes, and then flow is performed.

Tips

Use 1 mM DEAB, and keep cell concentrations equal between your experimental and negative control samples.

Results Summary

Results are gated as FITC+ on a contour scatterplot. MDA-MB-468 are a great positive control cell line.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

The only method for Aldh activity quantification

The Bad

Expensive, need to order solid DEAB for higher concentration

The Bottom Line

Is the most well-accepted and quickest way to quantify stem cell activity via Aldh activity. Expensive on a per-sample basis.

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