Membrane Sterol Staining Protocol

Staining of membrane sterols was performed as previously described (36 (link), 62 (link)). Briefly, log-phase cultures grown in YPD were stained for 5 min with 5 μg/mL of filipin (Sigma) and washed with PBS. After washing cells were immediately visualized with an inverted Zeiss microscope. Fluorescence was calculated using Cell Profiler software. Additionally, fluorescent levels were quantified with a fluorescent plate reader (Bio-Tek). Fluorescence was measured at the range of 360 to 470 nm wavelength and normalized by OD₆₀₀.

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Winski C.J., Qian Y., Mobashery S, & Santiago-Tirado F.H. (2022). An Atypical ABC Transporter Is Involved in Antifungal Resistance and Host Interactions in the Pathogenic Fungus Cryptococcus neoformans. mBio, 13(4), e01539-22.

Publication 2022

filipin fluorescent plate reader

Cell Filipin Fluorescence Membrane Microscope Sterols

Corresponding Organization : University of Notre Dame

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Variable analysis

independent variables

Staining of membrane sterols

dependent variables

Fluorescence levels
Fluorescence intensity

control variables

Log-phase cultures grown in YPD
Filipin staining concentration (5 μg/mL)
Washing with PBS
Wavelength range for fluorescence measurement (360 to 470 nm)
Normalization by OD600

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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