MMV Pathogen Box, MMV Pandemic Response Box, and GSK TB-active libraries were screened against M. abscessus ATCC 19977 strain which constitutively expressed the luxCDABE operon (M. abscessus lux) at 10 μM in duplicate in 96-well flat-bottom plates in 7H9 complete media. The culture was grown to log phase (OD600 0.4–0.8) and diluted to OD600 0.005 (5x106 CFU/mL). 90 μL of culture was mixed with 10μL of compound. Plates were sealed with parafilm and incubated at 37°C for 48 hours. Plates had a column of 0.1% DMSO as negative controls (drug-free conditions) and 64 μg/mL of AMK as positive controls. Luminescence was measured with an Infinite F200 Tecan plate reader. % Luminescence relative to DMSO control was plotted using GraphPad Prism version 9. Compounds that decreased luminescence ≤ 10% were classified as primary hits. Primary hits were screened against M. abscessus ATCC 19977 at 10 μM in triplicate using REMA (see “Determination of MIC” below). Plates were setup as performed for the primary screen. Fluorescence was measured with an Infinite F200 Tecan plate reader. % bacteria viability relative to DMSO control was plotted using GraphPad Prism version 9. Compounds that decreased bacteria viability ≤ 10% were classified as confirmed hits. Confirmed hits were followed up with dose-response curves to determine the minimum inhibitory concentration (MIC).
Infinite f200
Manufactured by Tecan
Sourced in Switzerland, Austria, Japan, United States, Germany, Italy, Australia
The Infinite F200 is a multimode microplate reader that provides precise measurements for a wide range of applications in life science research. It features high-performance optics and sensitive detectors to enable accurate absorbance, fluorescence, and luminescence detection.
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Lab products found in correlation
Enhanced atp assay kit, by Beyotime (5 mentions)
Dual luciferase reporter assay system, by Promega (5 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (4 mentions)
96 well plate, by Greiner (4 mentions)
Crystal violet, by Merck Group (4 mentions)
Wst 1 reagent, by Roche (4 mentions)
H2dcfda, by Merck Group (4 mentions)
Cell titer one solution cell proliferation assay, by Promega (4 mentions)
Calcein am, by Thermo Fisher Scientific (3 mentions)
Luciferase assay system, by Promega (3 mentions)
Dcfh da, by Merck Group (3 mentions)
Mtt assay, by Thermo Fisher Scientific (3 mentions)
96 well microplate, by Thermo Fisher Scientific (3 mentions)
Epoch 2, by Agilent Technologies (3 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (2 mentions)
Hek blue detection medium, by InvivoGen (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Resazurin, by Merck Group (2 mentions)
Celltiter 96 aqueous one solution reagent, by Promega (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
465 protocols using infinite f200
1
High-throughput Screening for M. abscessus
2
Automated Microbial Growth Assay
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Optical density was collected using a Tecan Infinite F200 for 96-sample plates. Each well/sample was measured at five different positions and the median value was used as the OD measure. A background level was measured in each plate and was subtracted from the measured OD.
Growth assays were conducted using a Tecan Freedom Evo 2000 liquid handling station. The 200 μl 96 sample plate was incubated at 30°C, for >24 h with an automatic scheduled OD measurement in the Tecan Infinite F200 executed every hour (65 (link)).
A log-linear fit was applied to each consecutive set of 10 data points, and the minimal doubling time was determined by the fit with the highest slope among the fits that passed the 5% significance threshold (test for linear fit, Bonferroni-corrected).
Growth assays were conducted using a Tecan Freedom Evo 2000 liquid handling station. The 200 μl 96 sample plate was incubated at 30°C, for >24 h with an automatic scheduled OD measurement in the Tecan Infinite F200 executed every hour (65 (link)).
A log-linear fit was applied to each consecutive set of 10 data points, and the minimal doubling time was determined by the fit with the highest slope among the fits that passed the 5% significance threshold (test for linear fit, Bonferroni-corrected).
3
Mitochondrial Function Assays
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To evaluate the ROS level, mitochondrial extracts were reacted with respiration buffer (including 123 mM KCl, 2 mM KH2PO4, 2.5 mM malate, 20 mM HEPES, 1 mM MgCl2, 5 mM pyruvate, and 500 μM EGTA) and 25 μM 2′,7′-dichlorofluorescin diacetate (DCF-DA). Then, the reactants were incubated in the dark for 20 min at 4 °C and fluorescence was measured at 485 nm (excitation wavelength) and 535 nm (emission wavelength) using a fluorescence microplate reader (Infinite F200, Tecan) [66 (link)].
To measure the MMP level, the mitochondrial extract was reacted with MI buffer with 5 mM pyruvate, 5 mM malate, and 1 μM JC-1. The reaction was gently mixed and incubated in the dark for 20 min at 4 °C. Then, fluorescence was measured at 530 nm (excitation wavelength) and 590 nm (emission wavelength) using a fluorescence microplate reader (Infinite F200, Tecan) [13 (link)].
To measure the ATP level, the mitochondrial extract was evaluated using a commercial ATP kit (Sigma-Aldrich chemical Co., Milwaukee, WI, USA). The ATP level was assessed according to the manufacturer’s protocol and reactants were measured using a luminometer (Glomax®, Promega, Sunnyvale, CA, USA).
To measure the MMP level, the mitochondrial extract was reacted with MI buffer with 5 mM pyruvate, 5 mM malate, and 1 μM JC-1. The reaction was gently mixed and incubated in the dark for 20 min at 4 °C. Then, fluorescence was measured at 530 nm (excitation wavelength) and 590 nm (emission wavelength) using a fluorescence microplate reader (Infinite F200, Tecan) [13 (link)].
To measure the ATP level, the mitochondrial extract was evaluated using a commercial ATP kit (Sigma-Aldrich chemical Co., Milwaukee, WI, USA). The ATP level was assessed according to the manufacturer’s protocol and reactants were measured using a luminometer (Glomax®, Promega, Sunnyvale, CA, USA).
4
Measuring Intracellular ROS via DCFH-DA
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The 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used to measure intrinsic and intracellular ROS production similar as previously described [24 (link)]. In short, the intrinsic ROS production was measured by first cleaving the diacetate using NaOH. After neutralization, the Ni was added in final concentrations of 10, 25 and 50 μg Ni/mL in a black 96 well plate with or without HRP (horseradish peroxidase, final concentration 2.2 U/mL). Fluorescence was then measured (excitation 485 nm, emission 535 nm) using a plate reader (Tecan Infinite F200). For the intracellular ROS, BEAS-2B cells were seeded in black 96-well plates with transparent bottom and were after 24 h loaded with 20 μM DCFH-DA in HBSS (Hank’s buffered salt solution) for 30 min at 37 °C. Subsequently, cells were washed with HBSS and exposed to 1, 5 and 10 μg Ni/mL of Ni, NiO NPs and NiCl2. Tert-butyl hydroperoxide (TBP, 10 and 30 μM) was used as positive control. Fluorescence was recorded every 5 min over 2 h (excitation 485 nm, emission 535 nm) using a plate reader (Tecan Infinite F200) at 37 °C and ROS increase was calculated as mean slope per min and normalized to the unexposed control. Results are presented as mean values ±standard error of mean (SEM) of three independent experiments.
5
ClpB Disaggregation of Heat-Aggregated Proteins
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ClpB disaggregation activities were performed by following the disaggregation of heat-aggregated Malate Dehydrogenase (MDH) (0.5 μM, 30 min at 47°C) and urea-denatured recombinant firefly luciferase (0.2 μM, 30 min at 30°C) as described elsewhere [20 (link), 34 (link)]. Chaperones of F. novicida were used at the following concentrations: 1 μM ClpB (wild-type or its variants), 1 μM DnaK, 0.2 μM DnaJ, and 0.1 μM GrpE. Disaggregation reactions were carried out in a reaction buffer (50 mM Tris pH 7.5, 150 mM KCl, 20 mM MgCl2, 2 mM DTT) containing an ATP-regenerating system (2 mM ATP, 3 mM phosphoenolpyruvate, 20 ng/μl pyruvate kinase). MDH disaggregation was monitored by turbidity measurement at an excitation and emission wavelength of 600 nm (Tecan Infinite F200). Considering the initial MDH turbidity as 100%, data were calculated compared to the denatured MDH and shown in percentage. For luminescence, reactions were incubated at 23°C for 60 min, aliquots (5 μL) were removed at the times indicated and luciferase activity was determined by adding 50 μM luciferin (Promega) and measuring light output in a Tecan Infinite F200. Reactivation of luciferase was determined compared to a non-denatured luciferase control.
6
Quantifying Interleukin-8 and AKT Kinase Activity
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The IL-8 Human ELISA Kit (KHC0081; Thermo Fisher Scientific, Waltham, MA, USA) was employed to determine the amount of interleukin-8 in the culture medium; 50 µL of the culture medium was used. Absorbance at 450 nm was recorded by microplate reader Infinite F200 (Tecan, Seestrasse, Switzerland). AKT Kinase Activity Assay Kit (ab139436; Abcam, Cambridge, UK) was used for determining AKT kinase activity. The signal was developed according to the manufacturer’s instructions. Absorbance at 450 nm was recorded by microplate reader Infinite F200 (Tecan, Seestrasse, Switzerland).
7
Mitochondrial Reactive Oxygen Species and Membrane Potential
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To investigate the reactive ROS level in mitochondria, mitochondrial extract was reacted with a respiration buffer including 125 mM KCl, 2 mM KH2SO4, 2.5 mM malate, 20 mM HEPES, 1 mM MgCl2, 5 mM pyruvate, 500 μM EGTA, and 25 μM DCF-DA. Then, the reactant was incubated in a dark room for 20 min, and fluorescence was measured using a fluorometer (Infinite F200, Tecan) at 485 nm (excitation wavelength) and 535 nm (emission wavelength) [29 (link)].
To measure the mitochondrial MMP level, the mitochondrial extract was reacted with MI buffer with 5 mM pyruvate, 5 mM malate, and 1 μM JC-1, and gently shaken. Then, the reactant was incubated in a dark room for 20 min, and fluorescence was measured at 530 nm (excitation wavelength) and 590 nm (emission wavelength) using a fluorometer (Infinite F200, Tecan) [30 (link)].
The ATP level was measured using an ATP kit (Sigma-Aldrich Chemical Co.) according to the manufacturer’s protocol. The reactant was then measured with a luminescence meter (GloMax, Promega, USA).
To measure the mitochondrial MMP level, the mitochondrial extract was reacted with MI buffer with 5 mM pyruvate, 5 mM malate, and 1 μM JC-1, and gently shaken. Then, the reactant was incubated in a dark room for 20 min, and fluorescence was measured at 530 nm (excitation wavelength) and 590 nm (emission wavelength) using a fluorometer (Infinite F200, Tecan) [30 (link)].
The ATP level was measured using an ATP kit (Sigma-Aldrich Chemical Co.) according to the manufacturer’s protocol. The reactant was then measured with a luminescence meter (GloMax, Promega, USA).
8
Quantifying Fluorescent Protein Expression
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GFP- and RFP-positive 293T cells were observed using an inverted fluorescence microscope (IX71, Olympus, Japan) and quantified via fluorometry (Infinite F200, TECAN). Transfected cells were observed using Axiovert™ software connected to a fluorescent microscope (magnification: 100×). GFP and RFP intensity were measured 24 h after transfection via fluorometry (Infinite F200, TECAN) according to the manufacturer's protocols. Relative GFP expression levels were determined by normalizing the values against RFP expression levels.
9
Quantifying Systemic Inflammation via Serum IL-6
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To evaluate systemic inflammation, 2 mL of blood was collected in a standard serum vial, incubated at 25 °C for 30 min, and left at 4 °C for 16 h to coagulate. The clot was centrifuged at 1200× g for 30 min. The supernatant was removed and stored at −20 °C. A Rat IL-6 enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen BMS625; Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to determine the serum levels of interleukin-6 (IL-6) according to the manufacturer’s instructions. The absorbance of each well was measured using an absorbance meter (Infinite® F200; Tecan Trading AG, Männedorf, Switzerland; primary wavelength, 450 nm; reference wavelength, 620 nm). A standard curve was created from the mean absorbance of each standard concentration, which was used to convert absorbance values to concentrations (pg/mL) (Excel 2016; Microsoft, Redmond, WA, USA).
10
Quantifying DNA using PicoGreen Assay
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Quant-iT™ PicoGreen dsDNA Assay Kit (Life Technologies; Cat. No. P7589) was used to quantify DNA by fluorescence. Lambda DNA contained in the kit was used to create a six-point standard curve from 3.125 to 100 ng/mL. DNA samples with a concentration determined by 260 nm absorbance higher than 100 ng/mL were diluted and later corrected through the dilution factor. Two microliters of each DNA and standard curve dilution were aliquoted into a CORNING 96 Flat Black plate (Corning, Inc.; Cat. No. 3650). 1× Tris-EDTA (TE) buffer was used as negative control. PicoGreen reagent was diluted 1:200 in 1× TE buffer and 198 μL was added to each well. Samples were mixed and incubated 15 minutes in darkness before their fluorescence was measured with the Infinite F200 instrument (Tecan Trading AG).
To estimate DNA integrity, the ratio between extraction yields in micrograms calculated using PicoGreen and spectrophotometry was determined. For tissue sections, yield was normalized for different samples using the nuclear area in square millimeters examined through hematoxylin staining. In brief, nuclei were counted in the microscope and the area occupied in square millimeters was estimated using a graticule. The total micrograms of DNA obtained for each sample was divided by the estimated area.
To estimate DNA integrity, the ratio between extraction yields in micrograms calculated using PicoGreen and spectrophotometry was determined. For tissue sections, yield was normalized for different samples using the nuclear area in square millimeters examined through hematoxylin staining. In brief, nuclei were counted in the microscope and the area occupied in square millimeters was estimated using a graticule. The total micrograms of DNA obtained for each sample was divided by the estimated area.
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