250 and 500 cells respectively for MCF-10a and MDA-MB-231 were seeded in triplicates in a 96 well plate. The number of cells was determine using CellTiter 96® Aqueous one (Promega, USA) cell proliferation assay solution at 24, 48, 72, 96 and 120 hours after seeding. Briefly, 20 μl of the reagent was added to each well and incubated at 37°C for 1 hour. The absorbance reading was taken at 490 nm and plotted in the graph as shown.
Celltiter 96 aqueous one
Manufactured by Promega
Sourced in United States
The CellTiter 96 AQueous One is a colorimetric assay solution used to measure the viability and proliferation of cells in culture. It contains a tetrazolium compound and an electron coupling reagent, which together measure the metabolic activity of cells. The assay provides a quantitative indication of the number of viable cells in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Small molecule inhibitors, by LC Laboratories (3 mentions)
384 well clear bottom plates, by Santa Cruz Biotechnology (2 mentions)
Dasatinib, by Selleck Chemicals (2 mentions)
Nilotinib, by Selleck Chemicals (2 mentions)
Imatinib, by Novartis (2 mentions)
Spark multimode microplate reader, by Tecan (2 mentions)
Calcusyn software, by Biosoft (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Microplate reader, by Tecan (1 mentions)
Synergy htx, by Agilent Technologies (1 mentions)
Quanti bluetm reagent, by InvivoGen (1 mentions)
Infinite 200, by Tecan (1 mentions)
Synergy neo2, by Agilent Technologies (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
Murine scf, by Thermo Fisher Scientific (1 mentions)
Ml141, by Merck Group (1 mentions)
Nsc23766, by Santa Cruz Biotechnology (1 mentions)
Rhosin, by Merck Group (1 mentions)
Zcl278, by Bio-Techne (1 mentions)
Np 40, by Merck Group (1 mentions)
56 protocols using celltiter 96 aqueous one
1
Cell Proliferation Assay for MCF-10a and MDA-MB-231
2
Synergistic Cell Killing Assay
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Cell viability was measured by MTS assay according to manufacturer’s instructions (CellTiter 96 AQueous One, Promega, Madison, WI). For testing synergistic cell killing, 786-O cells in 96-well plates (50,000 cells per well) were infected in sixtuplicate with serial dilutions of vaccinia virus and adenovirus, and 72 hours later viability was assessed by MTS and combination index calculated using CalcuSyn software (Biosoft) according to the method of Chou and Talalay.
3
Determining Cell Proliferation and Invasion in LnCaP and PC3 Cells
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LnCap or PC3 cells were seeded at 5 × 103 cells/well of a 96-well plate and incubated at 37 °C in 5% CO2 for 24 h before transfection with control scramble RNA or c-Jun siRNA. Proliferation was determined using the CellTiter 96 Aqueous one (Promega) assay. Briefly, 10 μL of the reagent was added to each well and incubated at 37 °C for 1 h in a humidified, 5% CO2 atmosphere, whereupon the absorbance reading was taken at 490 nm (Tecan Micro plate reader). For invasion assays, PC3 cells were seed in 10 cm dishes and transfected with control scramble RNA or c-Jun siRNA. After 24 h, the cells were trypsinized, washed, resuspended in serum-free RPMI-1640, and 1 × 105 cells plated in the upper chamber of 8-μm pore transwell chambers coated with 20 μg Matrigel. The lower chamber was filled with RPMI-1640 supplemented with 10% FBS as a chemoattractant. Following incubation at 37 °C for 24 h, non-migrated cells on the upper surface of the insert membrane were removed with a cotton swab. Cells on the lower side of membrane were removed by washing with PBS, and the CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used to determine the number of viable cells. All experiments were performed in triplicate and repeated three times.
4
Cell Viability Assay of HuCCT1 Cells
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HuCCT1 cells were transfected with siRNA in 96-well plate and incubated for 12 h. The medium was replaced with RPMI-1640 supplemented with 10 % FBS and further incubated for 24, 30 and 36 h followed by the addition of CellTiter 96 Aqueous One solution (Promega, WI). The reaction was incubated for 2 h and the absorbance at 490 nm was measured by Synergy HTX multi-mode reader (BioTek, VT). The assay was performed in three independent sets of triplicate wells.
5
BCR-ABL1 Inhibitor Cytotoxicity Assay
6
Bacterial Supernatant Effects on Reporter Cells
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For each experiment, reporter cells were seeded at 30,000 cells per well, into 96-well plates and incubated for 24 h. Then, cells were stimulated for 24 h with 10 µL of tested bacteria supernatant or resuspended bacterial pellet, for a final volume of 100 µL per well (i.e., 10% vol/vol), in the presence or absence of TNF-α (10 ng/mL final). Secreted Embryonic Alkaline Phosphatase (SEAP) in the supernatant was revealed using the Quanti-BlueTM reagent (Invivogen, Toulouse, France) according to the manufacturer’s protocol and quantified at 655 nm OD. All measurements were performed using a microplate reader (Infinite 200, Tecan, Lyon, France). Cell viability was controlled using an MTS assay (CellTiter 96 Aqueous One, Promega, Charbonnières-les-Bains, France) following the manufacturer’s instructions.
7
Cell Viability Assay with Inhibitors
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Small-molecule inhibitors, purchased from LC Laboratories (Woburn, MA, USA) and Selleck Chemicals (Houston, TX, USA), were reconstituted in DMSO and stored at −80 °C. Inhibitors were distributed into 384-well plates prepared with a single agent/well in a 7-point concentration series ranging from 10 μM to 0.0137 μM for each drug. The final concentration of DMSO was ≤0.1% in all wells; plates were stored at −20 °C and thawed immediately prior to use. Primary mononuclear cells were plated across inhibitor panels within 24 h of collection. Cells were seeded into 384-well assay plates at 10,000 cells/well in RPMI-1640 media supplemented with fetal bovine serum (10%), L -glutamine, penicillin-streptomycin and β-mercaptoethanol (10−4 M). After three days of culture at 37 °C in 5% CO2, MTS reagent (CellTiter96 AQueous One; Promega Madison, WI, USA) was added, optical density was measured at 490 nm [47 (link)].
8
Cytotoxicity Screening of Compounds
9
Measuring Growth Rates During p150 Depletion
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To measure growth rates during p150 depletion, HeLa S3 Trex-p150shRNA-1 and HeLa S3 Trex-shRNA-luciferase cells were plated at 1500 cells/well in 48-well plates. Triplicate wells for each time point (0–120 h) were assayed with CellTiter 96 AQueous One (Promega) per manufacturer's instructions. For growth analysis of cells expressing V5-tagged p150 or luciferase transgenes, 9000 cells/well in six-well dishes were infected with lentiviruses expressing either shRNA-luciferase or p150shRNA-1 at a multiplicity of infection (MOI) of 2. After 48 h, drug selection for infected cells was begun (1 mg/ml G418 or 0.5 μg/ml puromycin, respectively). After 7 d postinfection, cells were stained with crystal violet (Campeau et al., 2009 (link)) and photographed.
10
Drug Sensitivity Assay Protocol
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For drug sensitivity assays, 10,000 viable cells were dispensed into each well of a 384-well plate containing 7 point, threefold dilution, drug concentration series from a library of small molecule inhibitors. Cells were incubated with the drugs in RPMI media containing 10% FBS without supplementary cytokines. After 3 days of culture at 37 °C in 5% CO2, MTS reagent (CellTiter96 AQueous One; Promega) was added, the optical density was measured at 490 nm, and raw absorbance values were adjusted to a reference blank value and then used to determine cell viability (normalized to untreated control wells). Ex vivo functional drug screen data processing was performed as described, and dose response curve-fitting was carried out using the probit regression on quality-controlled data as in our previous work [3 (link)].
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