The quantitative analysis of biofilm formation was performed as reported previously13 (link) with minor modifications. Overnight cultures of Vibrio cholerae were diluted 1:100 in LB media supplemented with either 1% DMSO or 100 μM (S)-sebastenoic acid. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 100 μM when indicated. 100 μL of the diluted culture were grown in polyvinyl chloride (PVC) 96 well plates with “U” bottom (Corning) for 10 hours at 30°C under static conditions. A negative control of cell-free LB media was analyzed under the same conditions. The grown cultures (planktonic cells) were transferred to clear 96 well flat bottom plates (Corning) and the Optical Density at 595 nm (OD595) was measured using a Perkin Elmer Victor X3 plate reader. Each well was washed with sterile water to remove the remaining planktonic culture. 100 μL of 1% crystal violet (CV) was added to each well and incubated for 20 minutes at room temperature. CV was discarded and the wells were washed thoroughly with water. The wells were dried overnight and then 150 μL of 95% Ethanol were added. The CV adhered to the biofilms was solubilized by pipet mixing. 100 μL of each solution was transferred to clear 96 well flat bottom plates (Corning) and OD595 values measured using a Perkin Elmer Victor X3 plate reader. Six independent biological replicates were analyzed.
Clear flat bottom 96 well plate
Manufactured by Corning
Sourced in United States, United Kingdom
The Clear Flat Bottom 96-Well Plate is a laboratory equipment product designed for a variety of applications. It features a clear, flat-bottom construction that allows for optimal optical clarity and visibility. The plate contains 96 individual wells, providing a standardized format for conducting multiple experiments or assays simultaneously.
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Lab products found in correlation
Spectramax i3x minimax 300 imaging cytometer, by Molecular Devices (4 mentions)
Celltiter glo 3d cell viability assay, by Promega (4 mentions)
Poly 2 hydroxyethyl methacrylate, by Merck Group (4 mentions)
Y 27632, by Selleck Chemicals (4 mentions)
Triglyceride reagent, by Horiba (3 mentions)
Spectramax i3x plate reader, by Promega (2 mentions)
Matrigel, by Corning (2 mentions)
Growth factor reduced matrigel, by Corning (2 mentions)
D300e digital dispenser, by Tecan (2 mentions)
Spectramax i3, by Molecular Devices (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
No taurocholate, by Merck Group (2 mentions)
Synergy plate reader, by Agilent Technologies (2 mentions)
Sodium taurocholate, by Merck Group (2 mentions)
Cell counting kit 8 (cck8), by Merck Group (2 mentions)
Infinity cholesterol liquid stable reagent, by Thermo Fisher Scientific (1 mentions)
Softmax pro version 5, by Molecular Devices (1 mentions)
Vmax microplate reader, by Molecular Devices (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Woundmaker, by Sartorius (1 mentions)
40 protocols using clear flat bottom 96 well plate
1
Quantitative Biofilm Analysis of Vibrio cholerae
2
Small Molecule Inhibitor Screening for Sarcoma Cells
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ICR-SS-1 (2000/well), HS-SY-II (3000/well), and SYO-1 (2000/well) cells were seeded in clear 96-well, flat-bottom plates (Corning Inc., Corning, NY, USA). Plates were incubated for 24 h before replacing media with a panel of small molecule inhibitors at a concentration of 500 nM for all drugs except NVP-AUY922, which was at a concentration of 50 nM (details and source of inhibitors are shown in Supplementary Table S1 ). After 72 h, cell viability was determined using CellTitre-Glo (Promega, Madison, WI, USA), following the manufacturer’s instructions. Dose response assays were conducted by seeding ICR-SS-1 (2000/well), HS-SY-II (3000/well), and SYO-1 (2000/well) cells in clear 96-well, flat-bottom plates (Corning). Plates were incubated for 24 h, after which the medium was replaced with increasing concentrations of doxorubicin hydrochloride (Sigma Aldrich) or pazopanib (LC Laboratories) at the indicated dose. Data points from dose response assays were used to fit a four-point non-linear regression curve via Graphpad Prism and the drug screen data were subjected to hierarchical clustering using Perseus software [35 (link)] with Euclidean distance as the distance metric.
3
Serum Cholesterol Quantification
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Serum was initially diluted 1:10 in saline and 25 μL of diluted serum or standard was then added to a 96 well clear flat bottom plate (Costar) containing 100 μL of Infinity Cholesterol Liquid Stable Reagent (Thermo Scientific). Standards (Thermo Scientific) were prepared according to manufacturer’s instructions. Serum cholesterol was quantified at 500–520 nm using Molecule Devices vmax and Kinetic Microplate Reader and SoftMax Pro v5 software.
4
Hepatic Triglyceride Quantification Protocol
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Hepatic TG levels were quantified as described.14 , 17 , 18 Briefly, standards and samples were added to a 96‐well, clear, flat‐bottom plate (Costar) containing 200 μL of triglyceride reagent (Pointe Scientific). Hepatic TGs were quantified at 500‐520 nm using the vmax Microplate Reader (Molecular Devices) and SoftMax Pro version 5 software.
5
Hepatic Triglyceride Quantification in Mice
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A pre-weighed quantity of frozen mouse liver tissue was placed in 10 μL of Homogenization Buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF) per mg of liver tissue. A 1/8” steel bead was added to each sample, and samples were next dissociated using a TissueLyser (Qiagen) for 3 minutes at 30 Hz. Following 20 minutes of incubation on ice, each sample was diluted 1:10 in homogenization buffer and added to a 96 well clear flat bottom plate (Costar) containing 200 μL of Triglyceride Reagent (Pointe Scientific). Standards (Pointe Scientific) were prepared according to manufacturer’s instructions. Hepatic triglycerides were quantified at 500–520 nm using Molecule Devices vmax and Kinetic Microplate Reader and SoftMax Pro v5 software.
6
Serum Triglyceride Quantification Assay
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10 μL of serum was added to a 96 well clear flat bottom plate (Costar) containing 200 μL of Triglyceride Reagent (Pointe Scientific). Standards (Pointe Scientific) were prepared according to manufacturer’s instructions. Serum triglycerides were quantified at 500–520 nm using Molecule Devices vmax and Kinetic Microplate Reader and SoftMax Pro v5 software.
7
Quantifying Bacterial Growth Kinetics
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A single bacterial colony was inoculated into 5 mL of LB broth and grown overnight at 37°C at 250 rpm. The following morning, the bacteria was sub-cultured 1:100 in fresh LB for 3 h and then adjusted to an OD600 of 0.1 in LB broth. Volumes of 200 µL/well were added to a 96-well clear, flat-bottom plate (Corning, Corning, NY, USA). The plate was incubated at 37°C with shaking, and bacterial growth was quantified using a SpectraMax iD3 Multi-Mode Microplate Reader to measure OD600 every 30 min over 24 h. The R package Growthcurver (31 (link)) was then used to determine the growth rate from each growth curve. The number of replicates performed for each experiment is indicated in the figure legend showing these results.
8
Caco-2 Wound Healing Assay with CD8+ Supernatants
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Enriched CD8+ were co-cultured with THP1 cells loaded with fixed E. coli at 25 BpC in the presence or absence of 20 μg/mL LEAF anti-MR1 Antibody (Biolegend). Supernatants were collected at 72 hours. A total of 1.5x104 Caco2 cells were seeded per well in a 96-well clear flat bottom plate (Corning) and grown to confluency at 37°C for 5 days with media exchange every 2 days. Monolayers were scratched using a WoundMaker (Essen Bioscience), washed with serum-free medium and incubated with CD8+ 72h hour supernatants diluted 1:4 with fresh media. As a negative control, fresh media was used. Time lapse imaging was recorded every 4 hours using IncuCyte S3 Live Cell Analysis System (Essen Bioscience) for 36 hours at 37°C. GlutaMAX medium supplemented with 10% FBS, 1% NEAA, Penicillin-Streptomycin and L-glutamine was used throughout this experiment.
9
KGDH Enzyme Activity Assay
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The KGDH enzyme activity kit was purchased from Sigma (MAK189). Sample processing and activity assays were carried out as per kit instructions. Briefly, 1 × 106 cells were pelleted and washed once in PBS. Cells were then lysed in assay buffer for 10 min on ice in normoxia or hypoxia before being clarified by centrifugation at 4 °C for 5 min at 10,000g. The supernatant was collected, aliquoted, and snap frozen in liquid nitrogen before being stored in −80 °C for future use. On the day of the assay, samples were thawed on ice and aliquoted into a 96-well clear flat bottom plate (Corning). Kit-provided enzyme-specific developer and substrate reaction mixes were added to each sample, and the plate was then placed into a Cytation 5 instrument (BioTek). Absorbance was measured at 450 nm every minute at 37° for up to 2 h. Measurement analysis was then calculated as described in kit instructions.
10
Wound Healing Assay with H69 Cells
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Human cholangiocyte cell line H69 were cultured in 96-well plates. A total of 2 × 104 H69 cells were seeded per well in a 96-well clear flat bottom plate (Corning) and grown to confluency at 37 °C for 2 days and then scratched and washed with PBS before supplementing with different supernatants. Supernatants were collected at 5 days of sorted MAIT cells and diluted with RPMI medium containing 2% FBS in a ratio of 1: 3. Time lapse imaging was recorded every 4 h using microscopy (CKX53, Olympus, Tokyo, Japan) for 24 h. For anti-AREG blocking, 1 μg/mL of the polyclonal anti-AREG antibody was added to the supernatants. The open wound area were analyzed using ImageJ. The percentage of wound healing is defined as the quotient of the initial wound area and the wound area after a fixed time interval.
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