Disruption of mitochondrial function in S. cerevisiae Measured in Microorganism System Using Flow Cytometry - 2148-08_Inhibitor_SinglePoint_DryPowder_Activity
Assay Overview: A fluorescent dye (JC-1) will be used in a commercially available assay to quantitate the ability of test compounds to disrupt the mitochondrial membrane potential of fungi, at concentrations relevant to their previously determined anti-proliferative activity. This cationic dye exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~525 nm) to ..more
BioActive Compound: 1
Keywords: Saccaromyces cerevisiae, glycolysis, respiration, mitochondria
Assay Overview: A fluorescent dye (JC-1) will be used in a commercially available assay to quantitate the ability of test compounds to disrupt the mitochondrial membrane potential of fungi, at concentrations relevant to their previously determined anti-proliferative activity. This cationic dye exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~525 nm) to
yellow-red (~590 nm). Consequently, mitochondrial depolarization is indicated by a decrease in the yellow/green fluorescence intensity ratio.
Results will establish whether inhibition of growth in glycerol is
due to direct compromise of mitochondrial function or to other components of respiratory metabolism in fungi. This assay will be used for initial binning of hits based on mode of action: both probes that disrupt mitochondrial function as well as probes that perturb other components of respiratory metabolism will be of interest.
Expected Outcome: either increase or no change in signal
6.7 g/L Yeast Nitrogen Base (BD catalog # 239210)
0.79 g/L CSM amino acid supplement (Sunrise science products #1001-010)
2% Glucose (final concentration; added after autoclaving)
To 1L volume with H20.
Saccharomyces cerevisiae strain W303-1a (ATCC# 208352)
Positive control compound:
Antimycin A (use at 10 micromolar)
1. Grow a 2 mL overnight culture of W303 strain at 30C.
2. Dilute to an OD600 of 0.1 in 2 mL of fresh media (one tube per compound replicate).
3. Incubate at 30C with agitation for 90 minutes.
4. Add compounds at desired concentrations.
5. Incubate 4 hr at 30C with agitation.
6. Aliquot 1 mL of culture into a microcentrifuge tube.
7. Add JC-1 dye stock to tube at a final concentration of 1 ug/ml.
8. Incubate 30 minutes in the dark with agitation at 30C.
9. Wash out dye by centrifuging and resuspending in SD-CSM media twice.
10. Analyze by flow cytometry using a 488nM excitation laser and detect emission on FL1 and FL2 channels. Polarized mitochondria will emit strongly at ~590nM in the FL2 channel; while depolarized mitochondria will emit mainly in the FL1 channel. The ratio of FL2 to FL1 fluorescence thus reports on polarization, which must be initially calibrated with positive and negative controls.
The activity score column was calculated as follows:
The average score for the positive control, Ilicicolin H, was set as 100% active.
The excel formula "=ROUND(100-(100*(A-B)/C),0)" was used, in which A is the average value for the sample of interest, B is the average value for the positive control, and C is the average value for the negative control (DMSO).
Categorized Comment - additional comments and annotations
Data Table (Concise)