Fluorescence Cell-Based Secondary Assay for toxicity in mammalian fibroblasts
Assay Overview: The basic assay strategy will consist of NIH 3T3 mammalian fibroblasts cultured in 384-well format in the presence of a sub-toxic concentration of fluconazole. Test compounds that do not inhibit subsequent growth in the presence of fluconazole will merit further evaluation for their non-toxicity. This whole cell phenotypic screening approach will only capture compounds that are more ..
BioActive Compounds: 427
Keywords: Candida albicans, drug resistance, fluconazole, Hsp90, Calcineurin, stress response
Primary Collaborators: Susan Lindquist, Whitehead Institute for Biomedical Research, email@example.com
Assay Overview: The basic assay strategy will consist of NIH 3T3 mammalian fibroblasts cultured in 384-well format in the presence of a sub-toxic concentration of fluconazole. Test compounds that do not inhibit subsequent growth in the presence of fluconazole will merit further evaluation for their non-toxicity. This whole cell phenotypic screening approach will only capture compounds that are capable of entering and accumulating in cells to bioactive concentrations. Compound activity will be measured by the metabolism of Alamar Blue, a cell stain that is metabolized to a fluorescent product by living cells but that remains non-fluorescent in wells with growth-inhibited organisms.
a. Compounds that inhibit yeast growth in the presence, but not in the absence of 8 ug/ ml fluconazole.
b. Compounds that show a 10-fold specificity between the primary Candida test strain and mammalian cells.
c. Compounds that exhibit 10X greater inhibition against fungus than the hsp90 chaperone and /or calcineurin.
d. IC50 < 1uM
Expected Outcome: Wells in which there is toxicity will show a reduced fluorescence intensity due to a reduction in the amount of Alamar Blue dye metabolized by fewer viable cells.
Geldanamycin (GA) 15mM stock solution in DMSO
Pen/Strep 100x in PBS
Optimem medium (Invitrogen Cat#31985-070, Lot# 548536)
2.5% (v/v) Fetal Bovine Serum (Hyclone Cat#30071.03, Lot# ARF26748)
1% (v/v) Pen/Strep solution (Invitrogen Cat#15140-122, Lot#529891)
Test Strain: NIH-3T3 mammalian fibroblasts (ATCC# CRL1658)
Concentration in assay plate: Cells plated at 6,000/well in 20 uL assay medium and cultured overnight at 37C under 5% CO2 in 384-well, transparent bottom, black, tissue culture-treated, bar-coded assay plates
Procedure: After overnight culture, compounds are pinned into wells at 100nL/well using the CyBio pinning instrument. After compounds are pinned, an additional 20 uL of assay medium supplemented with fluconazole (16 ug/mL, Sequoia Research) is added to each well. The final nominal concentration in the well will be 25 uM of test compound and 8 ug/mL fluconazole. The plates will be returned to the tissue culture incubator and culture continued for 48 hrs at 37C under 5% CO2. At the completion of this incubation, Alamar Blue solution diluted 1:40 will be added to each well (10uL/well) to achieve a final dilution of 1:200. Plates will be incubated for a further 2-3 hrs at 37C under 5% CO2 and then fluorescence intensity as a measure of relative viable cell number will be determined on an Envision plate reading fluorometer.
Dilute Alamar Blue Reagent (Biosource International Cat# DAL1100) 1:40 in Ca/Mg-free PBS. Add 6.4 ul/well to plates with Combi (final dilution factor 1:200). Incubate 2 hrs at RT. Read Relative Fluorescence Intensity (RFU) of wells on standard plate reader as measure of relative fungal growth.
Plate reader set-up- Envision, Perkin Elmer: Ex 544nm, Em 590nm, Bandwidth 12nm, Top read.
Normalization of all wells was performed using the 'Neutral Controls' one-point normalization method in Genedata Assay Analyzer, where the median of the neutral control well signals is set to normalized value of 0, and relative inhibition values of compound wells are set to the percentage of the 0 signal value. Plate pattern correction algorithms were not used.
IC50 values were calculated using the curve fitting algorithms of Genedata
Screener Condoseo (v7.0.3). EC50 values were extrapolated up to 1 log
over the highest tested concentration.
Inactive compounds = 0
Active compounds = -10*Log(EC50)
Activity_Outcome = 1 (inactive)
EC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active)
EC50 <= 1 log over the highest tested concentration
Activity_Outcome = 3 (inconclusive)
Curve fitting strategy resulted in a constant fit with activity >30% but <70%
The fit was not valid due to poor fit quality.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)