Assaying Fungal Growth With XTT Assay

October 12, 2016

UW-Madison
Pediatrics
Postdoc

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Company:

ThermoFisher (Molecular Probes)

Product Name:

XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide)

Catalog Number:

X6493

To quantify fungal killing in vitro by leukocyte population you could use CFU, but for fungi that become hyphal like Candida albicans, plating for CFU can be inaccurate because of the biofilms formed in culture. I and others have used the XTT assay to quantify the amount of living C. albicans cells. Here I show a titration curve of C. albicans cells in a 96-well plate format. The XTT assay is an easier and effective way to track the survival of C. abicans in vitro. See Nett et al. 2011, doi: 10.1128/JCM.02273-10 for protocol reference.

Experimental Design and Results Summary

Application

In vitro viability assay (colorimetric)

Starting Material

Candida albicans cells (fungus)

Protocol Overview

Candida albicans strain 5314 was grown overnight at 30C in YPD (yeast extract, peptone, dextrose) medium with orbital shaking. Yeast were moved to RPMI+2% FCS to mimic killing assays with neutrophils (see Xu et al. 2016, http://www.ncbi.nlm.nih.gov/pubmed/27653689) and incubated at 37C. RPMI was washed from wells and wells were filled with 90 μl of XTT reagent (1 mg/ml XTT, 2% glucose in PBS) and 10 μl of 0.32 mg/ml phenazine methosulfate (also ThermoFisher) followed by a 30 minute incubation at 37° C before reading the absorbance at 450nm with a 96-well plate reader.

Tips

Data shown is an optimization step suggested before running a killing assay.

Results Summary

The XTT assay shows a linear increase in color change detected by absorbance as the number of C. albicans cells increases.

Additional Notes

None

Image Gallery

Summary

The Good

Much easier and faster than tracking CFU, especially for Candida

The Bad

Requires optimization for any type of fungal killing assay

The Bottom Line

The XTT assay is probably the best assay tested for tracking killing of Candida albicans by immune cells in vitro.

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