Bacterial cytotoxicity was assayed by adding bacterial supernatant extracts to Vero (African green monkey kidney) tissue culture monolayers. Bacterial supernatant extracts were prepared by centrifugation (13 000 X g, 5 min) of overnight cultures grown in CB supplemented with 0.2% sodium formate, 0.3% sodium fumarate, and 10% FCS. Supernatants were filtered through 0.2 μm membrane filters (Millipore Corporation, Bedford, MA). Vero cell monolayers were prepared in 96-well microtitre plates (Corning, USA) with Dulbecco’s Modification of Eagle’s Medium (DMEM) containing L-glutamine (Gibco Ltd) and 5% Fetal Bovine Serum (FBS), with incubation in 5% CO2 at 37°C for 24 h. Supernatant filtrates were added to Vero cells and incubated for 2 days at 5% CO2 at 37°C; the negative control was phosphate-buffered saline (PBS). Cells were assessed for cytotoxicity by looking for rounding of cells.
96 well microtitre plate
Manufactured by Corning
Sourced in United States
96-well microtitre plates are a type of laboratory equipment used for various assays and experiments. These plates consist of a grid of 96 individual wells, each capable of holding a small volume of liquid sample. The standardized well size and arrangement facilitate high-throughput screening, sample preparation, and data collection across multiple samples simultaneously.
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Lab products found in correlation
0.2 μm membrane filter, by Merck Group (1 mentions)
Calprotectin, by R&D Systems (1 mentions)
Microvue cic c1q eia, by Quidel (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by BD (1 mentions)
Rhmpo, by R&D Systems (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Anti mpo antibody, by Bio-Rad (1 mentions)
Dibucaine, by Merck Group (1 mentions)
Microplate reader, by Tecan (1 mentions)
Streptavidin horseradish peroxidase, by Bio-Synthesis (1 mentions)
Tetramethylbenzidine tmb substrate, by Tiangen Biotech (1 mentions)
Prism 6, by GraphPad (1 mentions)
Cis aconitate, by Merck Group (1 mentions)
Thermo multiskan ex micro plate photometer, by Thermo Fisher Scientific (1 mentions)
Fluoroskan ascent fluorescence spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
13 protocols using 96 well microtitre plate
1
Bacterial Cytotoxicity Assay on Vero Cells
2
Quantification of Circulating NETs by MPO-DNA ELISA
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Plasma levels of calprotectin (R&D Systems, Minneapolis, MN, USA) and immune complexes (ICs) (MicroVue CIC-C1q EIA, Quidel, Athens, OH, USA) were measured by ELISA, following the manufacturers’ instructions. The IC ELISA is based on the capacity of complement factor C1q, immobilized to the plate, to bind to circulating ICs. Quantification of circulating NETs was performed by utilizing myeloperoxidase (MPO)–DNA ELISA as described by us [10 (link)]. First, 96-well microtitre plates (Corning Inc., Corning, NY, USA) were coated with anti-MPO antibody (4 μg/ml; Bio-Rad Laboratories, Hercules, CA, USA) overnight at 4°C, and then blocked with 1% BSA in PBS for 2 h at room temperature (RT). Then, plasma samples diluted 1:100 (MPO–DNA ELISA) were added in 1% BSA in PBS with 2 mM EDTA, and incubated overnight at 4°C. Anti-DNA–HRP from the Cell Death Detection ELISA kit (clone MCA-33; Roche, Indianapolis, IN, USA) was added as detection antibody for 2 h at RT. The reaction was developed with 3,3′,5,5′-tetramethylbenzidine (BD Biosciences, San Jose, CA, USA) for 20 min and stopped by the addition of 2 M sulphuric acid. Known concentrations of MPO–DNA complexes (rhMPO, R&D Systems, Minneapolis, MN, USA; calf thymus DNA, Trevigen, Gaithersburg, MD, USA) were utilized to construct a standard curve. Absorbance was measured by a plate reader at 450 nm (Synergy 2, BioTek, Winooski, VT, USA).
3
Dibucaine Sensitivity Assay on LB Agar
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Sensitivity to the membrane perturbant dibucaine was assessed on LB agar plates. Briefly, 4-ml cultures were inoculated with overnight cultures at 1:100 dilution and grown in LB at 37°C until an OD600 of about 0.5 was reached. Cell counts were normalised according to OD600, then serially diluted in LB with seven 10-fold dilutions using 96-well microtitre plates (Corning). Two microlitres of the diluted cultures were manually spotted onto the LB agar plates and incubated overnight at 37°C. When indicated, dibucaine (Sigma-Aldrich) was added to the LB agar plate at a final concentration of 1.2 mM.
4
Antimicrobial Activity of Resazurin Compounds
5
ELISA for Detecting rhPHB Antibodies
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ELISA with rhPHB protein was performed as described previously [25 (link)]. Briefly, 0.1 µg/ml of rhPHB protein was coupled covalently to 96-well microtitre plates (Corning, NY). The plates were then blocked with 200 µl of 5% goat serum for 2 hours at 37°C. 100 µl of sera at 1 : 100 dilution was added and incubated for 2 hours at 37°C. Plates were then washed five times with 0.1% PBST. Then 100 µl of horseradish peroxidase-conjugated mouse anti-human IgG diluted at 1 : 15,000 was added. Bound antibodies were detected with tetramethylbenzidine (TMB) A (0.1 M citric acid, 0.2 M Na2HPO4, 0.6 g hydroperite/l) and TMB B (5 mM citric acid, 0.4 mM EDTA-Na2, and 0.2 g TMB/l) as substrate. Finally, the reaction was stopped by adding 2 M H2SO4. The absorbance at 450 nm was measured with a microplate reader (Tecan, Hombrechtikon, Switzerland).
6
Quantification of R97-116 Peptide Antibodies
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A standard ELISA technique was used to detect R97–116 peptide specific antibody. Briefly, R97–116 peptide (5 μg/ml) was coated onto a flat-bottomed 96-well microtitre plates (Corning) in 0.1 M carbonate bicarbonate buffer (pH 9.6) overnight at 4 °C. Then, the plates were blocked with 200 μl of PBS containing 0.05 % Tween20 and 10 % FCS at 37 °C for 1.5 h. A total volume of 100 μl diluted serum samples (1:100 in PBS/0.05 % Tween20) were added and incubated for 2 h at room temperature. After washed, biotin rabbit anti-rat IgG (1:3000; Biosynthesis Biotechnology, Beijing, China), biotin anti-rat IgG1, IgG2a, and IgG2b (1:500; BioLegend) were added to the wells and incubated for 1 h. Then streptavidin-horseradish peroxidase (1:1000; Biosynthesis Biotechnology) was added, incubated at 37 °C for 30 min. Then, plates were washed with PBS containing 0.05 % Tween20 and followed by development with Tetramethylbenzidine (TMB) substrate (Tiangen Biotechnology, Beijing, China). Finally, plates were read at a wave length of 450 nm using a microplate ELISA reader. Each serum was tested in triplicate. Results are expressed as mean OD value of samples ± SD.
7
Cytotoxic Effects of Cisplatin and LfcinB
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The in vitro cytotoxic effects of cisplatin or LfcinB on CAL27cis and Detroit‐562cis were tested in a standard MTT assay. For drug IC50 detection, CAL27cis or Detroit‐562cis in 96‐well microtitre plates (Corning, Bibby Sterilin Ltd, Staffordshire, U.K.) were treated with different dosages of cisplatin or LfcinB. After the incubation period of 24 hours, a standard MTT assay was performed as described in detail previously.4 IC50 was calculated using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). The cytotoxic effect of cisplatin or LfcinB was expressed as the relative viability (% of control) and was calculated as follows: Relative viability = (experimental absorbance − background absorbance)/(absorbance of untreated controls‐background absorbance) × 100%.
8
Enzymatic Activity Assay Protocol
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Cis-aconitate (CA, SIGMA) was dissolved in water and the pH was adjusted with NaOH to pH 7.4. MUG solution: 352 mg/l 4-Methyl-Umbelliferyl-beta-D Glucuronide (Promega/Serva) was dissolved in lysis buffer consisting of 328 ml/l 1 M Tris-HCl (pH 8), 120 mg BSA, 24 ml/l 10% SDS, 648 ml/l H2O. Resazurin (SIGMA) was dissolved in water (12.5 mg/100 ml). 96-well microtitre plates were from Corning.
9
Quantifying Carbohydrates and Uronic Acids
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Total carbohydrate content of NSP fractions was assayed using a modified method of Dubois et al.[47] . Briefly, 10 µL fractions were added to 96-well microtitre plates (Corning/Costar) in triplicate, and 100 µL of 4% (wt/vol) phenol dissolved in deionised water was added at room temperature for 5 min. Concentrated sulphuric acid (150 µL) was then rapidly delivered to all wells (with great care) and vigorously aspirated to generate heat required for reaction colour development. Plates were left to cool for 20 min and then measured for A560. Carbohydrate content of samples was determined using a calibration curve of d -glucose (0–20 µg/mL). Hexuronic acid content (d -glucuronic acid and d -galacturonic acid) was measured using a commercial K-URONIC assay (Megazyme International; Bray, Ireland). Increase in absorbance at 340 nm was determined upon incubation of fractions or 0–150 µg of d -glucuronic acid with uronate dehydrogenase in the presence of nicotinamide adenine dinucleotide (NAD+) at 25°C for 10 min, as per manufacturer's instructions.
10
Biofilm Formation Assay for T6SS Mutants
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Biofilm formation assays were performed on T6SS mutants of the Aaa strain RS-2 in 96-well microtitre plates (Corning-Costar Corp., Corning, NY, USA) using the method of Peeters et al. [48 (link)]. Briefly, the overnight cell suspension of the Aaa wild-type strain RS-2 and mutants were re-cultured into a fresh LB broth containing appropriate antibiotics with a 1:100 dilution under shaking to mid exponential growth. Then each well was inoculated with 100 µL of approximately ~1 × 108 CFU/mL (OD600 = 0.6) bacterial suspension and incubated at 30 °C for 48 h of adhesion without agitation while twelve wells filled with sterile ddH2O served as blanks. Culture media were then poured out and each well in the plates was washed three times with sterile ddH2O. Following air-dried for 30 min, each well was stained with 125 µL of 0.1% (w/v) crystal Violet (CV) solution for 45 min at room temperature. The unbound crystal Violet was removed and then washed with ddH2O. To solubilize the crystal Violet stained cells, 150 µL of 33% acetic acid was added into each well. Bacterial biofilm was quantified by measuring their optical density at 590 nm using a Thermo Multiskan EX Micro plate Photometer (Thermo Fisher Scientific Inc.). Twelve replications of each treatment were used for quantitative measurement in the three repeated experiments.
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