Cell viability following drug treatment was assayed in parallel with luciferase assays using CellTiter-Glo 2.0 (G9241, Promega) per manufacturer instructions and the BioTek Synergy 2 plate reader was used to evaluate the cytotoxic effect of the drugs. EC50s were calculated using GraphPad Prism.
Synergy 2 plate
Manufactured by Agilent Technologies
The Synergy 2 plate is a multi-mode microplate reader designed for a variety of applications in life science research. It is capable of performing absorbance, fluorescence, and luminescence measurements. The Synergy 2 plate offers a flexible platform for researchers to conduct assays and analysis in a high-throughput manner.
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9 protocols using synergy 2 plate
1
Cell Viability Assay for Drug Efficacy
2
Screening Antibiotic Compounds Against B. cenocepacia
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One hundred compounds selected from the primary screen were reassayed at a final concentration of 50 μM and 5% vol/vol DMSO in 96-well format. B. cenocepacia K56-2 was cultured overnight in LB broth, diluted to an A600 of 0.018 in LB and 95μl of the cell suspensions were added into 96 well plates containing the compounds to be tested in four replicates. Positive and negative control wells consisted of LB with DMSO (5% v/v) with or without bacterial cell suspensions, respectively. All plates were sealed, incubated at 37°C for 5 h, and after incubation, A600 was recorded after shaking for 15 seconds in a BioTek Synergy 2 plate reader. For each assay plate the residual growth was determined as the A600 in the presence of the tested compound/A600 in the absence of the compound.
3
Quantifying Oxidative Stress in Bacteria
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Superoxide anion was measured in prodrug and TPP treated bacteria by diluting them 1/50 into PBS containing 2 μM dihydroethidium (DHE) (Sigma Cat # D7008) just before flow cytometry (Excitation 535nm, Emission 610nm). H2O2 production was measured in similar bacterial samples using the Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit (Thermo Fisher Cat # A22188). Fluorescence measurements were calibrated by comparison to calibration curves for wells containing H2O2 in a final concentration ranging between 0.1 to 100 μM. Fluorescence was measured using the 540/620 nm wavelength pair in a Biotek Synergy 2 plate reader.
4
Cell Viability Quantification using CellTiter-Glo
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Following quantification of toxicity, the number of viable cells/well was quantified by adding 25 μl CellTiter-Glo reagent to each well. To maximize cell lysis plates were mixed on an orbital shaker (700 to 900rpm) for 5 min before equilibration at room temperature for an additional 10 min. Quantitation was achieved by measuring luminescence per well using a Biotek Synergy 2 plate reader.
5
Quantitative Pyocyanin Extraction and Measurement
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Pyocyanin was extracted from the supernatants of liquid cultures by adding an equal volume of chloroform (CHCl3) and vigorously vortexing. The lower, pyocyanin-containing organic layer was then taken and vortexed with an equal volume of 0.2 M HCl. The pink pyocyanin-containing aqueous layer was taken, and its absorbance at 520 nm (A520) was read in a BioTek Synergy 2 plate reader.
For some experiments, pyocyanin was quantified directly in culture supernatants by reading the absorbance at 691 nm as previously described [19] (link). Sterile medium was used as a blank.
For some experiments, pyocyanin was quantified directly in culture supernatants by reading the absorbance at 691 nm as previously described [19] (link). Sterile medium was used as a blank.
6
RNA Isolation, cDNA Synthesis, and qRT-PCR Analysis
7
Enzyme Inhibition Kinetics Assay
8
Chelator-mediated regulation of Histoplasma growth
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Histoplasma yeasts from exponentially growing cultures were collected, washed, and resuspended in RPMI medium containing serial dilutions of the copper chelator bathocuproinedisulfonic acid (BCS), the iron chelator bathophenanthrolinedisulfonic acid (BPS), or the zinc chelator N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) or, alternatively, in HMM medium containing supplemental copper(II) sulfate. Histoplasma cultures were incubated at 37°C with continuous shaking (200 rpm), and yeast growth was quantified by measuring the culture turbidity (optical density at 595 nm) with a BioTek Synergy 2 plate reader. The Histoplasma growth in each medium is reported relative to the growth in basal medium alone.
9
Quantifying Oxidative Stress in Bacteria
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