Black flat bottom 96 well microplates

Peptide deformylase catalyzes the removal of the N-formyl group from formyl-Met-Ala-Ser. The free amino group reacts with fluorescamine to form highly fluorescent products which can be monitored with a TECAN Infinite M1000 PRO multi-mode microplate reader by exciting at 390 nm and emission at 470 nm. For screening, assays were performed in black flat-bottom 96-well microplates (Corning). First, 49.5 μl reaction solution (20 nM VaPDF, 1 mM formyl-Met-Ala-Ser and 25 mM HEPES, pH 7.4) was dispensed in each well and then 0.5 μl dimethylsulfoxide (DMSO) or samples dissolved in DMSO (4 mg/ml) was dispensed. Plates were incubated at 37°C for 30 min. Then fluorescamine was added to a final concentration of 60 μg/ml. The fluorescence intensity (FI) of each well was detected. The inhibitory values were calculated as (FI_sample-FI_{negativecontrol})/(FI_{positivecontrol}-FI_{negativecontrol}) × 100%.
Dimethylsulfoxide was chosen as negative control and heat-inactivated VaPDF as positive control during measurements. The Z′ factor and CV values were calculated as follows:
Z′ = 1–3(SD_FImax-SD_FImin)/(Mean_FImax-Mean_FImin), SD: standard deviation. The theoretical value is between 0.5 and 1. CV(%) = SD_FImax/Mean_FImax or CV(%) = SD_FImin/Mean_FImin. The acceptable value of CV for HTS assay is less than 10%.

Ethidium bromide uptake was measured as previously described (27 (link)). M. tuberculosis mc²7000 and derivative strains were grown to mid-exponential phase (OD₆₀₀ of 0.4 to 0.6), pelleted by centrifugation, washed once with PBS-T, and resuspended in PBS-T to an OD₆₀₀ of 0.4 to 0.5. Ethidium bromide was added at a 2-μg/ml final concentration, and uptake was measured using black, flat-bottom, 96-well microplates (Corning) and a Synergy H1 Hybrid plate reader (BioTek) in top-reading mode with excitation at 544 nm and emission at 590 nm. Uptake rates were determined using data in the linear range between 0 and 30 min and are the mean values ± standard deviations of at least three independent experiments.

3D scans were performed on a Shimadzu RF 6000 spectrofluorophotometer. Enzyme activity by UV, fluorescence measurements and OD₆₀₀ determinations were performed using a Tecan Spark 10 M microplate reader. Enzyme activity characterization through conversions was performed by HPLC measurements, using an Agilent Technologies 1200 Series instrument equipped with autosampler. The UV irradiation of assay samples was performed in Corning 96-well Clear Flat Bottom UV-Transparent microplates using a handheld Analytik Jena UV lamp of 302 nm. The fluorescence measurement was carried out in Corning 96-well Black Flat Bottom microplates. Styrene was purchased from Sigma Aldrich and solvents (n-hexane, n-octane, dimethyl sulfoxide, acetonitrile) of HPLC grade were purchased from VWR. The synthesis of fluorogenic probe 4 was performed according to reported procedure^{47 (link)}.