C. albicans chitinase activity was quantified from isolates with LBF (n = 3) and HBF (n = 3) after 4 and 24 h of biofilm formation using a fluorometric chitinase assay kit (Sigma, United Kingdom), as per the manufacturer’s instructions. Following biofilm development, supernatants were collected at 4 and 24 h, and an appropriate volume of each sample was incubated with a substrate working solution (4-methylumbelliferyl N-acetyl-β-d-glucosaminide) at 37°C for 30 min. Fluorescence was then quantified at Ex360/Em450. Appropriate positive and negative controls included in the kit were added to each plate. Chitinase activity was calculated and expressed as a ratio between chitinase units and optical density of culture (U/OD). Each isolate was measured in duplicate, on three separate occasions.
Fluorometric chitinase assay kit
Manufactured by Merck Group
Sourced in United Kingdom
The Fluorometric chitinase assay kit is a laboratory tool used to quantify the activity of the enzyme chitinase. Chitinase is an enzyme that breaks down chitin, a structural component found in the cell walls of fungi and the exoskeletons of insects and crustaceans. The kit utilizes a fluorescent substrate to measure chitinase activity, allowing for the accurate and sensitive detection of this enzyme.
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3 protocols using fluorometric chitinase assay kit
1
Quantifying C. albicans Chitinase Activity
2
Fluorometric Chitinase Assay Protocol
3
Fluorometric Chitinase Assay Protocol
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Cells were grown in MM for 15.5 h, reaching OD600 = 0.75-1.25. 5 ml culture samples were subject to centrifugation at 3,720g for 10 min to remove cells. Chitinase activity in the resulting supernatants was determined using a fluorometric chitinase assay kit (Sigma) with the synthetic substrate 4-methylumbelliferyl N,N’-diacetyl-β-D-chitobioside. Statistical analysis of the results was carried out using GraphPad Prism version 7.03 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com ). Normality of datasets was assessed by the Kolmogorov-Smirnov test. Normally distributed datasets were compared to the parental strain using one-way ANOVA followed by Dunn’s multiple comparison test. Non-normally distributed datasets were compared to WT using a Kruskal-Wallis test followed by Dunnett’s multiple comparisons test.
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