P131 Optimization of MTT assay protocol in Pythium insidiosum zoospores incubated together with neutrophils

POSTER SESSION 1, SEPTEMBER 21, 2022, 12:30 PM - 1:30 PM: OBJECTIVES: The tetrazolium-based MTT assay is widely used to assess cell viability. This colorimetric assay measures cell viability through the enzymatic reduction of MTT, a yellow water-soluble tetrazolium dye, to purple-colored formazan crystals. The zoospores of Pythium insidiosum are the infective form causing pythiosis. There remains a lack of standardized protocol for P. insidiosum zoospore viability assay. Here, we optimized the MTT assay to assess the activity of neutrophils on zoospores. METHODS: Neutrophils and zoospores were incubated for 2 h, 4 h, and overnight at 37°C. Neutrophils were lysed in water (pH = 11), MTT was added to each well and incubated at 37°C for 2 h. Then, SDS-HCl solution was added to each well, and absorbance at 570 nm was measured using a microplate reader. The percentage of viable zoospores was then determined. RESULTS: The viable zoospores metabolized MTT to insoluble purple formazan product. The zoospores germinated into hyphae when incubated for ˃2 h. Because of the rapid germination of zoospores into hyphae, reliable results would be obtained with an optimum incubation time of 2 h for the zoospores. CONCLUSION: The simplicity and low cost of the assay make it a valuable tool for zoospore viability studies, with a focus on immunology and immunotherapy.