A library of 2,476 FDA approved drugs was purchased from MedChem Express (Monmouth Junction, NJ, United States), and preliminary analysis was performed in a 96-well microtiter plate (Corning costar, Cambridge, MA, United States). P. aeruginosa PAO1 strain was used for high-throughput screening. Briefly, overnight cultures of P. aeruginosa PAO1 were suspended in MH broth to 1 × 106 CFU/ml. Hundred microliter of bacterial suspension was added to 96 μl of MH broth, and 4 μl of screening drugs from the drug library was added to wells of a 96-well microtiter plate, achieving a concentration of 100 μM. The plate was then incubated at 37°C for 24 h. After treatment, we used the turbidimeter to determine the survival rate of planktonic cells at an optical density (OD) of 630 nm (Campbell, 2011 (link); Sun et al., 2016 (link)) and classified the drugs that reduce the turbidity of culture by more than 50% as compared with the untreated control as the plate was gently washed with 1 × PBS after the culture was removed. The inhibition rate of biofilm formation was determined by measuring at 570 nm of 0.5% (w/V) crystal violet dissolved in ethanol. Experimental assays were performed twice. The second screening reduced the drug concentration to 30 μM. The screening procedure was the same as the initial screen. Experimental assays were performed in triplicate.
96 well microtiter plate
Manufactured by Corning
Sourced in United States, United Kingdom, Germany, China
The 96-well microtiter plates are a common laboratory equipment used for various experimental and analytical applications. These plates consist of a rectangular array of 96 individual wells, typically arranged in a 8x12 format. The wells are designed to hold small volumes of liquids, samples, or reagents, enabling parallel processing and high-throughput experimentation.
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Lab products found in correlation
Microplate reader, by Thermo Fisher Scientific (6 mentions)
Elisa plate reader, by Bio-Rad (5 mentions)
Microplate reader, by Agilent Technologies (4 mentions)
Celltiter glo, by Promega (4 mentions)
Microplate reader, by Bio-Rad (4 mentions)
Wst 8, by Roche (4 mentions)
Imark microplate absorbance reader, by Bio-Rad (4 mentions)
Prism 6, by GraphPad (3 mentions)
Dnase 1, by Merck Group (3 mentions)
Spectramax id5, by Molecular Devices (3 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by BD (3 mentions)
Horseradish peroxidase linked anti mouse igg, by GE Healthcare (3 mentions)
Ysi poly l lysine coated spa beads, by PerkinElmer (3 mentions)
Cck 8, by Corning (3 mentions)
Microplate reader, by Molecular Devices (3 mentions)
Cck 8, by Roche (3 mentions)
Hts tubulin polymerization assay kit, by Cytoskeleton (2 mentions)
Versamax microplate reader, by Agilent Technologies (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Mueller hinton broth (mhb), by Beijing Land Bridge Technology (2 mentions)
259 protocols using 96 well microtiter plate
1
High-Throughput Screening of FDA-Approved Drugs Against Pseudomonas aeruginosa
2
Quantification of Bacterial Biofilm Formation
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Twenty-four-hour cultures were diluted with TSB medium (containing 0.5% glucose) at a ratio of 1:200, inoculated into a 96-well microtiter plate (Costar, USA), and incubated statically at 37°C for 6, 12, 24 and 48 h. The bacterial culture was discarded, and nonadherent cells were washed three times with PBS. The biofilm was fixed with 99% methanol and stained with 2% crystal violet for 15 min. The culture plates were rinsed and dried at room temperature. Finally, OD570 values were determined by a SpectraMax 190 Microplate Reader (MD, USA). Experiments were repeated at least three times (Zhu et al., 2017 (link)).
3
AgrC Inhibition Peptidomimetic Assay
4
Serum IgE Quantification by ELISA
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Blood samples were collected by centrifugation at 2500 g for 20 min. The serum was diluted (1:250) with 5% fetal bovine serum (FBS) in PBS (assay diluent) and the serum IgE levels were measured using a mouse IgE ELISA kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, a 96-well microtiter plate (Costar, NY, USA) was coated overnight at 4 °C with anti-mouse IgE mAb. After wash with PBS containing 0.05% Tween 20, the plates were blocked with 5% FBS in PBS for 1 h at RT. The diluted 100 µL serum samples were incubated for 2 h at RT. Secondary peroxidase-labeled biotinylated anti-rat IgE mAb was incubated in blocking buffer for 1 h. The enzyme reaction was initiated by addition of TMB substrate solution (BD Biosciences) for 30 min, and the reaction was stopped by the addition of 50 µL stop solution to each well. The optical density was measured at 450 nm with a microplate reader (SOFT max PRO, version 3.1. Molecular Devices, Sunnyvale, CA, USA). The lower limit of detection for the IgE ELISA was 1.5 ng/mL.
5
Norspermidine Biofilm Inhibition Assay
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Experiment was conducted based on the method previously described by Santiago and Lim (Santiago et al. 2015a ,b ). A 96‐well microtiter plate (Corning/Costar, USA) was prepared with norspermidine at the following concentrations: 0, 2, 2.5, 3, 3.5, and 4 mmol/L. Aliquots of PAO1 suspension (adjust to 0.5 McFarland) were added to these wells. The plate was incubated for 2 h at 37°C. Following incubation, the wells were washed with saline and biomass of cell attachment was determined by crystal violet staining method as described earlier.
6
Sialidase Activity Assay Using 4-MUNANA
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Sialidase activity was assayed by using 4-MUNANA (Sigma) as the substrate as previously described (4 (link)). Lysates of transfected cells were suspended in 200 μl of 50 mM sodium acetate buffer (pH 4.4) containing 0.1% Triton X-100, protease inhibitor cocktail (Sigma), and 25 μl of 4-MUNANA (2 mM). The mixtures were briefly vortexed and incubated for 1 h at 37°C. For the 4-MUNANA assay, the reaction was terminated with 1 ml of glycine buffer (pH 10.3) containing 0.133 M glycine, 60 mM NaCl, and 42 mM Na2CO3. After the mixtures were centrifuged, the supernatants were collected and dispensed into a 96-well microtiter plate (Costar) to measure the fluorescence intensity by Fluoroskan Ascent FL (Thermo Electron Corporation). The amount of hydrolyzed 4-MUNANA in each sample was interpolated with the intensities from a serial dilution of a known concentration of 4-MUNANA (Sigma) with GraphPad Prism 4 (GraphPad Software, Inc., La Jolla, CA). In experiments using antibody immunoblockade or pharmacological inhibition, the cell lysates in the reaction buffer were incubated with heat-inactivated immune serum or inhibitor at the dilution indicated for 15 min at room temperature prior to substrate introduction.
7
Tubulin Polymerization High-Throughput Assay
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The Tubulin Polymerization HTS Assay Kit (Cytoskeleton, BK011P) was used according to the manufacturer's instructions. All components were added into a 96-well microtiter plate (Corning Costar, cat. no. 3686), then the tubulin reaction mixture was quickly added to the wells, and tubulin polymerization was initiated and monitored every 1 min at 37°C for 1 h by recording fluorescence of excitation wavelength at 340 nm and emission at 450 nm. The tubulin reaction mixture was composed of 80 mM PIPES (pH 6.9), and 1 mM MgCl2, 1 mM EGTA, 1 mM GTP and 2 mg ml−1 of highly purified porcine brain tubulin heterodimer (Cytoskeleton, cat. no. T240).
8
Biofilm Formation Assay for E. faecalis
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E. faecalis strains were inoculated in BHI with the indicated antibiotics and kept 16 hr at 37°C. The next morning, they were diluted in a 1:100 ratio in TSB-D with the indicated antibiotics, 10 ng/ml cCF10, and 50 ng/ml nisin. 200 µl fractions were dispensed into a 96-well microtiter plate (Costar) with 8 replicates per strain. 200 µl TSB-D fractions were used as blanks. The 96-well plate was then incubated aerobically at 37°C without agitation in a humidified chamber for 24 hr. The suspension was transferred to another 96-well plate to determine the optical density at 600 nm (OD600). The plate containing the biofilm was washed with distilled water three times and then left to air dry at room temp for 2.5 hr. The biofilm was stained with 100 µl 0.1% (wt/vol) safranin (Sigma) at room temp for 20 min, then washed three times with distilled water and left to air dry at room temperature. Afterwards the absorbance was determined using a plate reader (BMG Labtech) at 450 nm. Biofilm production was calculated as an index of safranin staining of the cell biomass divided by absorbance of its optical density (OD450/OD600) (Willett et al., 2019 (link)).
9
Biofilm Formation on Microtiter Plates
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The method of biofilm formation on the bottom of a 96-well microtiter plate (Costar Corning, New York, USA) was used according to described by Thein et al. [17 (link)]. Firstly, 100 μL of the standardized suspension of C. albicans was added to each well of the plate. The plate was incubated for 90 min at 37°C under shaking at 75 rpm (Quimis, Diadema, São Paulo, Brazil) to promote initial adhesion of the microorganisms. The suspension was then aspirated and each well was washed two times with PBS to remove weakly adhered cells. Next, 100 μL fetal bovine serum was added to each well and the plates were incubated again for 2 h under shaking. The wells were washed two times with PBS and 50 μL of the standardized S. mutans suspension or 50 μL of the S. mutans culture filtrate was added to each well. In the control groups, 50 μL of PBS (Control PBS) or 50 μL of BHI + 5% sucrose were performed (Control BHI). In addition, a control group formed only by 50 μL of the standardized S. mutans suspension (without C. albicans) was added.
For biofilm growth and maintenance, 140 μL yeast nitrogen base (YNB) supplemented with 100 mM glucose and 60 μL BHI broth supplemented with 5% sucrose were added in each well. The media were changed at intervals of 24 h and the plates were incubated at 37°C under shaking at 75 rpm for 48 h.
For biofilm growth and maintenance, 140 μL yeast nitrogen base (YNB) supplemented with 100 mM glucose and 60 μL BHI broth supplemented with 5% sucrose were added in each well. The media were changed at intervals of 24 h and the plates were incubated at 37°C under shaking at 75 rpm for 48 h.
10
SARS-CoV-2 RBD Protein Binding Assay
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A 96-well microtiter plate (Costar, USA) was coated with 0.5 μg per well of the recombinant SARS-CoV-2 RBD protein (Gene Universal, USA) in coating buffer (50 mmol/L Na2CO3, pH 9.6) and incubated overnight at 4 °C. Serum was diluted 200-fold with blocking buffer, added and processed as described previously (Jin et al., 2022 ). The results are reported as the positive index (ratio of sample OD450 to control OD450) (Andrianaivoarimanana et al., 2012 ; Phillips et al., 2018 ; Jin et al., 2022 ).
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