To assess metabolic activity by means of the generation of reduction equivalents, neutrophils were plasma treated and divided in aliquots immediately (9 replicates/sample) into clear, flat-bottom, tissue culturetreated, 96-well plates. Resazurin (20 ml) was added after 2, 4, and 6 h to 3 replicates, each containing neutrophils or HBSS alone for a blank value. Resazurin freely diffuses into the cells, where it is reduced via NADH/NADPHdependent reductase activity to its fluorescent form resorufin. Fluorescence (l ex 530 nm; l em 590 nm) was recorded continuously .30 min at 37°C using a plate reader (F200 Pro; Tecan, Switzerland). Background (resazurin alone) was subtracted and the slope for each sample calculated. For apoptosis, neutrophils were plasma treated and divided in aliquots immediately in black, 96-well plates, and caspase 3/7 detection reagent, which is cleaved specifically by these caspases to unmask a fluorescent DNA-binding dye, was added. Cells were incubated at 37°C, and fluorescence (l ex 485 nm; l em 535 nm) was recorded after 2, 4, and 6 h using a plate reader (F200 Pro; Tecan).
F200 pro
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The F200 Pro is a multifunction microplate reader designed for a wide range of applications in life science research and drug discovery. It features high-performance optical components and advanced detection technologies to deliver precise and reliable data across a variety of sample types and assay formats.
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Lab products found in correlation
Y1003, by Merck Group (2 mentions)
Pierce ldh cytotoxicity assay kit, by Roche (2 mentions)
Streptavidin donor bead, by PerkinElmer (1 mentions)
Alphalisa technology, by PerkinElmer (1 mentions)
Glutathione acceptor beads, by PerkinElmer (1 mentions)
Enduren, by Promega (1 mentions)
Cytotox glo, by Promega (1 mentions)
Daf fm da, by Beyotime (1 mentions)
Saponin, by Carl Roth (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Millicell ers voltohmmeter, by Merck Group (1 mentions)
Transwell plate, by Corning (1 mentions)
Cyclic amp xp chemiluminescent assay kit, by Cell Signaling Technology (1 mentions)
In cell analyzer 2200, by INCELL (1 mentions)
Ez rich defined medium, by Teknova (1 mentions)
Enduren live cell luciferase substrate, by Promega (1 mentions)
35 protocols using f200 pro
1
Neutrophil Metabolic Activity and Apoptosis
2
Screening for Dislocation Inhibitors
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We screened LOPAC small-molecule library (SigmaAldrich.com) for dislocation inhibitors using the HeLa cells stably expressing the NHK-drGFP reporter (Zhong and Fang, 2012 (link)). NHK-drGFP reporter cells were seeded at 2 × 104/well in a black wall, clear-bottom 96-well plate (3603; Costar.com) and cultured overnight. The next day, culture medium containing the proteasome inhibitor BTZ (1 µM) alone or together with each compound (10 μM) was added to the cells for an additional 4 h. Wells containing medium alone served as background control. After replaced the medium with 1× PBS, drGFP fluorescence intensity was measured on a TECAN F200 Pro multimode microplate reader using excitation = 488 nm and emission = 525 nm. Compounds that exhibited >70% inhibition compared with BTZ-alone samples after the background extraction were subject to measurement of auto green fluorescence, and fluorescent compounds were eliminated. The remaining compounds were retested in an NHK-drGFP assay and were denoted as dislocation inhibitors when confirmed. All dislocation inhibitors were further verified independently in the InCell Analyzer 2200 wide-field imaging system at the National Center for Advancing Translational Sciences (NCATS) using a different batch of compounds at 10 μM concentration.
3
Heterologous gene expression in E. coli
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Electrocompetent TG1 E. coli were transformed by electroporation and plated onto LB agar plates with the appropriate antibiotics. Single colonies were cultured in 2xYT media (Sigma‐Aldrich Y1003) with appropriate antibiotics, incubated at 37°C in a Stuart SI500 orbital shaker (220 rpm) and grown to stationary phase for ∼16 h. The culture was transferred to a 96‐well plate (Greiner 655090) and 10 µl of each inducer dissolved in 2xYT media (Sigma‐Aldrich Y1003) was added to a final volume of 150 µl. Measurements of absorbance at 600 nm, GFP and RFP fluorescence were taken with the Tecan f200pro multimode microplate reader with the following settings: 37°C, 16 h kinetic cycle with 10 min kinetic interval, orbital shaking (2 mm amplitude, 280 rpm frequency), absorbance at 595/10 nm, GFP excitation at 485/20 nm, GFP emission at 535/25 nm, GFP gain of 20, GFP mirror Dichroic 510 nm, RFP excitation at 590/20 nm, RFP emission at 625/35 nm, RFP gain of 38, RFP 50% mirror, fluorescence top reading, fluorescence integration time 20 μs.
4
Hsp90 Inhibitor Screening Assay
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The binding assays were performed using an Hsp90 (C-terminal) Inhibitor Screening Kits (cat. 50314 or 50317) purchased from BPS Bioscience. The assay was performed according to the manufacturer's protocol and utilised AlphaLISA® technology (PerkinElmer). The test compounds were dissolved in 100% DMSO and diluted with water to the desired concentration, so that the final dilution was dissolved in 5% DMSO with water. Two microlitres of the dilution was added to a 10 µL reaction so that the final concentration of DMSO was 1% in all reactions. The reactions were conducted at room temperature for 30 min in a 10 µL mixture containing assay buffer, 6 ng (24 nM) of a C-terminal Hsp90β (UniProt P08238, a.a. 527–724) or C-terminal Hsp90α (UniProt P07900, a.a. 535–732), 40 ng (60 nM) Cyp40, and the test compound. Note: for the full-length Hsp90 binding assays, C-terminal Hsp90 was replaced with 24 nM of full-length Hsp90α (Abcam cat # ab48801). After the 30 min incubation, 10 µL of buffer containing 20 µg/mL glutathione acceptor beads (Perkin Elmer) were added to the reaction mix and incubated for 30 min in the dark. Ten microlitres of 40 µg/mL streptavidin donor beads (Perkin Elmer) were then added and the final 30 µL mixture was incubated for 1 h in the dark. The AlphaLISA® signal was measured using a Tecan F200 Pro multimode plate reader.
5
Thioflavin T Fluorescence Assay for S100A9
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Thioflavin$T fluorescence assay was performed by adding 20 UM thioflavin$T to S100A9 solu$ ions kept on ice and then pipetted into 96$well plates. Thioflavin$T fluorescence was measured by a Tecan F200 Pro plate reader, using an excitation filter at 450 nm and an emission filter at 490 nm.
6
Pst Growth and Elicitor Screening
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After growth in King’s B medium, Pst cells were collected by centrifugation at 3000 g for 5 min and resuspended to a final optical density of 0.01 (OD600nm). Then corresponding elicitor or ethanol was added to this solution, which was subsequently distributed in 96 wells plate. OD600nm was monitored every hour during 48 h with a TECAN F200 pro. The bactericide effect of elicitor was monitored by counting CFU 24 h post-treatment. To this end, King’s B medium containing Pst supplemented with elicitor or ethanol was serial diluted in 10 mM MgCl2 and CFU/mL was counted on solid KB medium dishes supplemented with antibiotics.
7
Actin Polymerization Kinetics Assay
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The in vitro actin polymerization assay was carried out with Actin Polymerization Biochem Kit according to the manufacturer’s protocols. The solution of G-actin monomer (0.08 mg/ml) was incubated on ice for 1 h and centrifuged for 30 min, 14,000 rpm at 4 °C to depolymerize any remaining oligomers in solution. Modified AuNPs were added to 180 μl G-actin solution to make final concentration of 10 μg/ml. Actin polymerization buffer (500 mM KCl, 20 mM MgCl2, 0.05 M guanidine carbonate, and 10 mM ATP, 20 μl) was added to each well and the fluorescence signal was observed every 30 s for 1 h, using Tecan F200pro microplate reader (Männedorf, Switzerland). The experiment was carried out three times in triplicates.
8
Fluorescence Polarization Assay for BAX Binding
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Fluorescence polarization assays (FPA) were performed as previously described37 (link). Firstly, direct binding isotherms were generated by incubating FITC-BIM SAHBA2 (50 nM) with serial dilutions of full-length BAX and fluorescence polarization was measured at 20 min on a F200 PRO microplate reader (TECAN). Subsequently, in competition assays, a serial dilution of small-molecule or acetylated BIM SAHBA2 (Ac-BIM SAHB) was combined with FITC-BIM SAHBA2 (50 nM), followed by the addition of recombinant protein at EC75 concentration, as determined by the direct binding assay (BAX: 500 nM). EC50 and IC50 values were calculated by nonlinear regression analysis of competitive binding curves using Graphpad Prism software.
9
Fluorescence Polarization Assay for RAR-alpha Binding
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The fluorescein-tagged peptide of N-CoR1, FITC-Ahx-RLITLADHICQIITQDFAR (FITC-N-CoR1) was provided by Genscript at purity > 95%. Fluorescence polarization assays (FPA) were performed using established procedures36 (link). Direct binding isotherms were generated by incubating FITC-N-CoR1 (5 nM) with or without small molecules (10 μΜ) with serial dilutions of RARα LBD starting from 10 μΜ and diluted two-fold at each step. The buffer solution for assays was 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 5 mM DTT and 10% (v/v) glycerol. Fluorescence polarization was measured at 30 min on a F200 PRO microplate reader (TECAN) with the excitation wavelength set at 470 nm and emission measured at 530 nm. EC50 values were calculated by nonlinear regression analysis of competitive binding curves using GraphPad Prism software.
10
BRET Assay for Protein-Protein Interactions
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