For XBP1s induction experiments, tunicamycin was used at 1 μg/mL for the indicated periods of time. For inhibitor cell toxicity assays, cells were plated in 96 well plates at 5000 cells per well and treated with 0, 5, 10, 25, 50, 100, 250, 500, 1000 and 2500 μM concentrations of each inhibitor. After 6 days of incubation, WST1 reagent (Roche) was added to each well and post 2-hour incubation the plate was read using a Tecan 200 colorimeter. For TMZ sensitivity assays, cells were plated in a 96 well plate at 5000 cells per well and co-treated with 0, 5, 10, 25, 50, 100, 250, 500, 1000 and 2500 μM of TMZ plus a non-toxic dose of inhibitor. After 6 days of incubation, WST1 reagent (Roche) was added to each well and post 2-hour incubation the plate was read using a Tecan 200 colorimeter.
Wst 1 reagent
Manufactured by Roche
Sourced in Germany, Switzerland, United States, United Kingdom, Canada, France, Belgium, Australia, Italy, Spain
The WST-1 reagent is a colorimetric assay used for the quantitative determination of viable cells. It measures the activity of mitochondrial dehydrogenases, which convert the WST-1 substrate into a water-soluble formazan dye. The intensity of the formazan dye produced is proportional to the number of metabolically active cells in the sample.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Synergy ht, by Agilent Technologies (7 mentions)
Synergy ht microplate reader, by Agilent Technologies (7 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (7 mentions)
Wst 1 reagent, by Agilent Technologies (6 mentions)
Microplate reader, by Bio-Rad (6 mentions)
Crystal violet, by Merck Group (6 mentions)
Microplate reader, by Molecular Devices (5 mentions)
Infinite m200, by Tecan (4 mentions)
Dtx 880 multimode detector, by Beckman Coulter (4 mentions)
Wst 1 assay, by Roche (4 mentions)
Fluostar omega plate reader, by BMG Labtech (4 mentions)
Spectramax m5, by Molecular Devices (4 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (4 mentions)
Infinite f200, by Tecan (4 mentions)
Multiskan go, by Thermo Fisher Scientific (4 mentions)
Prism 6, by GraphPad (4 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Laminin, by Merck Group (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
492 protocols using wst 1 reagent
1
Inducing and Evaluating XBP1s Expression
2
Osteoblast Differentiation and Mineralization
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Cells were seeded onto a 12-well plate, with 1 × 104 MC3T3-E1 cells per well. OGFs (100 nM dexamethasone, 0.05 mM ascorbic acid 2-phosphate, and 10 mM b-glycerophosphate; Sigma-Aldrich, St. Louis, MO, USA) were added to trigger osteoblast differentiation under different treatments. After the cells had been treated, they were fixed in 60% citrate-buffered acetone and then incubated with a mixture of Fast Violet B Salt and naphthol AS-MX phosphate alkaline solutions (Sigma-Aldrich, St. Louis, MO, USA) for the detection of ALP expression. ALP activity and the total number of cells were measured using the TRACP & ALP assay kit (TakaRa Bio, Kusatasu, Shiga, Japan) and water-soluble tetrazolium salt (WST-1) reagent (Roche, Basel, Switzerland), respectively, in accordance with the manufacturers’ instructions. Cells were fixed with 10% paraformaldehyde after treatment, and then a 2% Alizarin Red S solution (ScienCell, Carlsbad, CA, USA) was used for detecting calcification. Calcium LiquiColor Assay (Stanbio Laboratory, Boeme, TX, USA) and WST-1 reagent (Roche, Basel, Switzerland) were respectively used in accordance with the manufacturers’ instructions to determine the calcium content and number of cells after treatment.
3
Cell Viability Assay for UVC-Exposed ARPE-19 and RCE Cells
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ARPE-19 and RCE (procured from ECACC) cells were grown as described in Supplementary data . After 2 h, plates were dried, exposed to UVC rays (60 mJ/cm2 for ARPE19 and 25 mJ/cm2 for RCE) in a Stratalinker 1800 apparatus (Stratagene, La Jolla, CA), and fresh medium containing the drugs was added. After 24 h (for ARPE-19 and RCE, respectively), cells were analyzed for viability by the WST-1 reagent (Roche Molecular Biochemicals) according to the manufacturer’s protocol: briefly, the medium was replaced with a complete RPMI 1640 medium without phenol red with 10% WST-1 reagent. Plates were incubated for 1.5 h at 37 °C in a 5% CO2, humidified atmosphere, vigorously shaken 1 min at room temperature, and analyzed by a microplate reader at 450 nm (Bio-Rad, Hercules, CA).
4
Capsaicin's Impact on Cell Growth
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In order to measure the effect of capsaicine on cell proliferation, cells were plated at a concentration of 1×104 cells/ml for in a 96 well plate overnight. Then cells were treated with DMSO and different concentrations of capsaicin (150, 200 µM) for different time points (1–4 days). At the respective time points, 10 µL WST-1 reagents (Roche Diagnostics, Mannheim) was added to each well and incubated for 2 h at 37°C. The absorbance was detected at a wavelength of 492 nm with reference wavelength of 650 nm.
5
Antibodies and Cell Reagents for Prion Research
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KA, glimepiride and phospholipase C were purchased from Sigma (Poole Dorset, UK). Fluoro-Jade B was from Millipore (Billerica, MA), SYBR green (Applied Biosystems, USA) and WST−1 reagents were from Roche (Basel, Switzerland). Lipofectamine plus was from Invitrogen (Carlsbad, CA). The following antibodies were used in this study: Anti-PrP SAF61 mouse monoclonal antibody (1:1000 diluted) antibody was from Spi-Bio (Cayman Chemical, Massy, France) and anti-PrP 6H4 mouse monoclonal antibody (1:5000 diluted western blotting and 1:250 immunocytochemistry) antibody was from Prionics (Schlieren, Switzerland). Mouse monoclonal antibody anti-tubulin (1:10000 diluted) was from Sigma (Poole Dorset, UK) and rabbit-raised polyclonal antibody against GFAP (1:500 diluted) was from Millipore (Billerica, MA).
6
LPS-Induced Cytotoxicity in RIMVECs
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RIMVECs (1 × 104 cells/well) were seeded in a 96-well plate and treated with a final concentrations of 1 μg/mL LPS for different time points (0.5, 1, 2, 4, 8, 12, and 24 h) at 37°C in a 5% CO2 atmosphere. After treatment, the cytotoxicity of RIMVECs was detected by 10 μL of WST-1 reagents (Roche). After 1 h incubation at 37°C, the absorbance was detected by a fluorescence microplate reader (Life Science & Technology) at wavelength of 450 nm. The percentage of RIMVECs survival was calculated based on the ratio of absorbance compared to DMEM treated group. After RIMVECs treated with LPS, then cells were washed with PBS and incubated with PI (5 μg/mL, Sigma-Aldrich) for 30 min. The PI positive cells presented the membrane damaged cells and fluorescence intensity of PI was immediately detected with excitation wavelength at 535 nm and emission wavelength at 615 nm.
7
Cell Viability Assay for OTS964
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In vitro cell viability was measured by the colorimeteric assay using WST-1 reagents (Roche Diagnostics, Indianapolis, IN, USA). U87 and U251 cells (1 × 103 cells/100 µl) were plated in 96-well plates. The cells were allowed to adhere overnight before exposure to various concentrations of OTS964 for 72 hours at 37°C. Plates were read with a spectrophotometer at a wavelength of 450 nm. All assays were carried out in triplicate. The values of IC50 were calculated by the straight line system made from 2 points of concentration of OTS964 around the 50% inhibition of cell viability.
8
V2O5 Nanoparticle Cytotoxicity Evaluation
9
Cell Proliferation and Migration Assays
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Cell proliferation and migration assays were performed as described previously [30] . For proliferation assay, a cell concentration of 5 × 10 3 cells/well was adopted for 48 h incubation. After the incubation period, 10 μl premixed WST-1 reagents (Roche Applied Science, Mannheim, Germany) was added into each well, and the cells were incubated for 4 h in a humidified atmosphere (e.g., 37℃ 5% CO 2 ). Afterwards, the absorbance of the samples was measured at 450 nm against a background control with a reference wavelength higher than 600 nm on a microtiter plate (ELISA) reader.
For migration assay, images were taken at 0, 6, 12, and 15 h, and superimposed using PhotoImpact (Adobe). The number of cells that migrated into the wounded space were manually counted in three fields per well under a light microscope at × 40 magnifications. Areas were quantified by using Image J analysis software.
For migration assay, images were taken at 0, 6, 12, and 15 h, and superimposed using PhotoImpact (Adobe). The number of cells that migrated into the wounded space were manually counted in three fields per well under a light microscope at × 40 magnifications. Areas were quantified by using Image J analysis software.
10
Inhibition of B Cell Proliferation
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Example 5
Mouse splenocytes were obtained by dissociating spleens of C57BL/6 mice on a 70 μm cell strainer and lysing red blood cell in RBC buffer (Sigma). B cells were then purified by depleting CD43-positive cells using magnetic microbeads (Miltenyi Biotec) according to manufacturer's instructions. The obtained B cells were subsequently stimulated with goat anti-mouse IgM antibody (Jackson Laboratories) at 10 μg/mL final assay concentration. Recombinant VNARs were serially-diluted and pre-complexed with recombinant BAFF-Fc at 5 ng/mL final assay concentration in RPMI 1640 supplemented with 10% FBS, for 30 minutes at 37° C. Stimulated B cells were added to the pre-complexed proteins and further incubated for 72 hours at 37° C., 5% CO2. Cell proliferation was then estimated by incubating cells with WST-1 reagent (Roche) and reading absorbance at 450 nM, subtracting a reference wavelength at 595 nM (
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