Human acute promyelocytic leukemia cells, NB4 (purchased from DSMZ company, Braunschweig, Germany) were cultured in RPMI 1640 Medium (Biological Industries, Beit-Haemek, Israel), supplemented with 10% of FBS (Biological Industries, Israel) and 1% of antibiotics (Biological Industries, Israel) at 37 °C in a humidified atmosphere of 5% CO2 in the air. The effects of new potential cytotoxic drugs on cell viability were determined by MTS assay as previously described [55 (link)]. Briefly, the cells were seeded in 96-well plates (3000 cells/well) and incubated over-night at 37 °C in 5% CO2. After 24 h, the cells were treated with compounds 1 −4 at different concentrations varied from 0.625 to 80 µM. As2O3 was used as a reference compound. After 8 h, 24 h and 48 h of incubation, 20 μL aliquots of MTS solution (CellTiter 96® AQueous reagent, Promega, Mannheim, Germany) were added to each well and re-incubated for 2 h at 37 °C. Then, the absorbance was measured at 492 nm, on microplate reader (Apollo-1, LB913, Berthold, Bad Wildbad, Germany). The percentage of cell viability was calculated from the control group. All treatments were conducted in sixplicate and the experiments were repeated thrice.
Celltiter 96 aqueous reagent
Manufactured by Promega
Sourced in United States
The CellTiter 96® AQueous reagent is a colorimetric assay used to determine the number of viable cells in a proliferation or cytotoxicity assay. It contains a tetrazolium compound and an electron coupling reagent, which together produce a colored formazan product that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Sartorius (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Victor3, by PerkinElmer (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
Hepg2 cell line atcc hb 8065, by American Type Culture Collection (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Multiscan fc, by Thermo Fisher Scientific (1 mentions)
Elx800 plate reader, by Agilent Technologies (1 mentions)
Imark microplate absorbance reader, by Bio-Rad (1 mentions)
Infinite m1000 pro, by Tecan (1 mentions)
Infinite f200, by Tecan (1 mentions)
14 protocols using celltiter 96 aqueous reagent
1
Cytotoxic Evaluation of Novel Compounds
2
Hydrogel-based 3D cell culture assay
3
Cell Viability Assay using CellTiter96
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Cultured cells were washed two times with PBS and then a small amount of the cellTiter96 AQueous reagent (Promega, Leiden, Netherlands) was added to the wells. The reagent was allowed to incubate for 2 h before recording the absorbance at 490 nm with a 96-wells plate reader (Perkin Elmer Victor 3, Waltham, MA, USA).
4
Regulation of YAP1 in Cancer Cells
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LNCaP GFP, LNCaP GFP-YAP1-WT, and LNCaP GFP-YAP1-Y407 were grown to 70–80% confluency in RPMI media. Cells were then treated with 10 µM J54 for 24 h. Thereafter, the treated cells were harvested for RNA extraction. For mitomycin C (Sigma-Aldrich, cat# M-0503, St. Louis, MO, USA) treatment, Hek293 cells were treated with 3 μM MMC for 24 h or as indicated. Treated cells were collected and processed for cell lysate for RNA extraction or immunoblotting. For the cycloheximide chase assay, 50 μg/mL of CHX (Sigma-Aldrich, cat# C-7698, St. Louis, MO, USA) was used as the final concentration. Cells were collected at indicated times and processed with a RIPA buffer for immunoblotting. The cell viability assay was performed using MTT as per the manufacturer’s instruction (Promega Corp., CellTiter 96® Aqueous reagent, cat# G3580, WI, USA).
5
Cell Viability Assay Using MTS
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The cell viability was determined using the MTS test. The cells were plated in 96-well microtiter plates (100 μL; TPP, Switzerland) at a concentration of 5,000 cells/well 1 day before the treatment for 24 h in the growth medium and then treated for 60 min in different hypotonic Leibovitz-water or sucrose-water solutions. Afterward, the hypotonic solution of interest was replaced by fresh growth medium in the cell culture for another 48 h, followed by the addition of MTS reagent (CellTiter 96 AQueous Reagent; Promega, United States) for 1 h. In the control measurements, the cells were treated for 60 min in either undiluted Leibovitz’s medium or an iso-osmolar sucrose-water solution. The absorbance, which corresponds to the amount of formed soluble formazan product that is directly proportional to the number of viable cells, was measured at a wavelength of 490 nm. The cell viability was determined as the ratio of the absorbance measured in the cells in different hypotonic solutions and the absorbance in the undiluted Leibovitz’s medium.
6
Characterization of Colagenart® Bioactive Compounds
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Three batches of Colagenart® (HC018‐01, HC018‐02, and HC018‐03) were provided by LEMAR S.A.P.I. de C.V. (Mexico City, Mexico). Sodium chloride, and monobasic, dibasic sodium phosphate, and sodium 3‐(trimethylsilyl) tetradeuteriopropionate (TSP) (analytical grade) were acquired from J. T. Baker; (NY, USA). Water, formic acid, and acetonitrile (MS grade) and dimethyl sulfoxide (DMSO) (analytical grade) were acquired from Sigma‐Aldrich (MO, USA). Note that 99.98% deuterated water solution was acquired from Cambridge Isotope Laboratories, Inc. (MA, USA). HepG2 cell line (ATCC® HB‐8065; VA, USA) and CaCo‐2 cell line (ATCC® HTB‐37) were acquired from ATCC (VA, USA). Fetal bovine serum and DMEM medium were obtained from Thermo Fisher Scientific (MA, USA). CellTiter 96® AQueous reagent was acquired from Promega (WI, USA).
7
Dehydrogenase Activity Assay Protocol
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The dehydrogenase activity was performed using the CellTiter96 AQueous reagent (Promega, Madison, WI, USA) according to the protocol provided by the manufacturer. After contact with the materials, 100 µL of bacteria cell suspension were mixed with 20 µL of the reagent and incubated at 37 °C for up to 4 h depending on the bacterial strain. After incubation, the absorbance was measured at 490 nm using microplate reader Multiscan FC (Thermo Fisher Scientific, Waltham, MA, USA). The results are presented as percent values using control as 100%.
8
MTS Assay for δT Antiproliferation
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A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was conducted to analyze the antiproliferative effects of δT on the above NSCLC cell lines; 5×104 of A549 and H1299 cells were grown in 96-well plates overnight. Afterward, the media were changed, and the cells were treated with fresh cell growth medium containing <0.01% DMSO (control) or treatment medium containing 10, 20, 30, and 40 μM of δT. After 72 hours of treatment, 20 μL of CellTiter 96® Aqueous Reagent from Promega (Madison, WI, USA) was mixed into each well and incubated for 2 hours. The absorbance at 490 nm was then measured using the Bio-Tek ELx800™ plate reader (Winooski, VT, USA). Each variant of the experiment was performed with six replicates.
9
Cell Viability Assay Protocol
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At various time points posttreatment, cells were collected by centrifuging. Next, collected cells were exposed to CellTiter 96® AQueous reagent (Promega, USA) and incubated at 37 °C for 2 h. The iMark Microplate Absorbance Reader (Bio-Rad) was used for detection of the absorbance of cells.
10
Cell Viability Assay with MI-401 and SD
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Cells (3,000/well) were seeded in a 96-multiwell plate (Corning Costar) with complete medium (100μL per well), and incubated at 37°C, with 5% CO2. The cells were then treated with various concentrations of MI-401 or SD (1–400 μM) for the indicated time periods. The MTS assays in a 96-well plate were done with a CellTiter 96® AQueous reagent (20 μl, Promega) in a culture medium (100 μl). The plates were incubated at 37°C for 4 h. Absorbance was measured at 490 nm using a microplate reader (Infinite M1000 Pro, Tecan, Männedorf, Switzerland). The experiments were done in at least triplicate.
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