Live/Dead Staining D. Discoidium with Calcein-AM

February 18, 2019

Princeton University
Molecular Biology
Graduate Student

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Company:

Invitrogen

Product Name:

Calcein, AM, cell-permeant dye

Catalog Number:

C3100MP

Our lab has tested the use of this dye as a reporter for cell death of the amoeba, D. discodium, a versatile model host for studying bacterial pathogenesis. We found that calcein-AM staining was an effective method for quantifying killing of D. discoidium by bacterial pathogens and purified bacteria-derived cytotoxins. Although calcein-AM is marketed as a cell-permeant dye that labels viable cells, we found that for D. discoidium, it is impermeable to living cells and only enters the cytoplasm of dying cells with compromised membranes. Staining is rapid, and produces a very strong signal compared to background autofluorescence.

Experimental Design and Results Summary

Application

Live/Dead Quantification of D. discoidium

Starting Material

D. discoidium

Protocol Overview

Live/Dead staining can be performed using a microscopy-based assay or a high-throughput, microplate-based assay. For microscopy: (1) Prepare pads for imaging - Dilute 1 mM stock solution of calcein-AM (in DMSO) 1:1000 into molten 1% agar prepared using desired D. discoidium growth medium. Let agar solidify on a glass surface, then cut into 1 cm x 1 cm squares. (2) Prepare samples - Add 1 µL of D. discoidium culture to a microscope slide, and add 1 µL growth medium with 2% hydrogen peroxide. Prepare a control sample without hydrogen peroxide. Cover samples with agar pad, and incubate for 30 min. Image cells using fluorescent microscopy in the phase and FITC channel. To quantify bacterial pathogenesis, add 1 uL of D. discoidium to a microscope slide that has been coated with a monolayer of a bacterial pathogen of interest, such as P. aeruginosa. Include a control with a relevant mutant or non-pathogenic strain. Follow directions as described above.

Tips

Make sure calcein-AM stocks and supplemented media are well mixed and do not contain particulates. Keep calcein-AM media in the dark to avoid bleaching.

Results Summary

After 30 min of incubation, the untreated D. discoidium are not stained with calcein-AM, while the peroxide-treated cells and all fluorescently labelled. Similarly, D. discoidium grown with pathogenic bacteria are stained, while those grown with a non-pathogenic strain are not (see figure).

DOI or PMID #

N/A

Additional Notes

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Image Gallery

Summary

The Good

It produces a strong signal with a broad dynamic range. There is little background fluorescence in living D. discoidium cells. The assay is very rapid, and cell death can be imaged immediately.

The Bad

It can degrade or bleach if not kept in the proper conditions. There can be background spots that would affect fluorescent quantification. Also quantification of mean fluorescence per cell can be variable. This product is idea for a binary live/dead assay.

The Bottom Line

I highly recommend this product for assaying cell death of this organism. It is versatile and easy to use.

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