Cell proliferation assays were performed using the CellTiter 96™ Aqueous Non-Radioactive Proliferation Assay (Promega Corp., Madison, WI) as described previously (14 (link)). This assay measures the reduction of a tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), by living cells to a formazan product. Briefly, the glioma cells, 1×105/ml in DMEM with 10% FCS, were plated in 96-well plates (Becton Dickinston, Lincoln Park, NJ) at 5,000 cells. After 24-h incubation, the various doses of IFN-β (10 to 5,000 U/ml) were added to the wells. The cells were incubated for 48, 96, 144 h. At the end of incubation period, to the microplate wells were added 20 μl of a freshly prepared combined tetrazolium compound and an electron coupling reagent (phenazine methosulfate) solution, then incubated for 2 h at 37°C, and the optical density at 490 nm was read on an automatic microplate reader (Model 550, Bio-Rad). The experiment was repeated at least three times in triplicate wells for each concentration of IFN-β.
Celltiter 96 aqueous non radioactive proliferation assay
Manufactured by Promega
Sourced in United States
The CellTiter 96™ Aqueous Non-Radioactive Proliferation Assay is a colorimetric method for determining the number of viable cells in proliferation assays. The assay quantifies the conversion of a tetrazolium compound into a colored formazan product that is soluble in tissue culture medium.
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Lab products found in correlation
Model 550, by Bio-Rad (1 mentions)
96 well flat bottom tissue culture plate, by BD (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Polarstar optima, by BMG Labtech (1 mentions)
Cytation 3 plate reader, by Agilent Technologies (1 mentions)
Prism 8, by GraphPad (1 mentions)
Infinite m200 microplate reader, by Tecan (1 mentions)
6 protocols using celltiter 96 aqueous non radioactive proliferation assay
1
Cell Proliferation Assay for IFN-β Treatment
2
Assessing lncRNA Expression Impact on Cell Proliferation
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UMSCC-10B, UMSCC-22B, HN-1, and HN-30 cells were plated into a 96-well flat-bottom tissue culture plate (Falcon) at a density of 5000 cells per well. After a 24-h plating period, cells were transfected with the lncRNA expression plasmids. Following a 48- to 72-h incubation period, cellular proliferation was analyzed using the CellTiter 96 AQueous nonradioactive proliferation assay (Promega) in accordance with the manufacturer's protocol. All assays were performed in triplicate wells and experiments were individually performed twice.
3
Live/Dead Staining and Quantitative Analysis
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Treatment efficacy was determined by in situ Live/Dead staining using calcein AM (ThermoFisher) and propidium iodide (Sigma–Aldrich), followed by fluorescence imaging (Olympus FV1000 confocal laser scanning microscope), as optimized for 3D cultures [26 (link)]. Derivation of outcome parameters was done using quantitative image analysis according to the CALYPSO methodology, as described previously [27 (link)]. When indicated, culture viability was assessed based on NAD(P)H oxidase activity using a CellTiter 96 aqueous non-radioactive proliferation assay (MTS, Promega, Madison, WI, USA), which was performed in accordance with the manufacturer’s recommendation.
4
Cell Viability Assay with MTS
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Cell viability was determined by using the Celltiter 96 AQueous nonradioactive Proliferation Assay (Promega). Cells were seeded in sterile 96-well plates at a density of 2000 cells per well and incubated for 72 h or 120 h at 37 °C and 5% CO2. Compound and vehicle were added to a final concentration of 0.5% DMSO. After 72 or 120 h of incubation time, 20 μL of a mixture (20 : 1) consisting of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-2H-tetrazolium) and PMS (phenazine methosulfate) were added to each well. Absorbance was measured after another 2–4 h with a BMG LABTECH POLARstar OPTIMA plate reader (BMG Labtechnologies, Germany). Experiments were performed in triplicates and GI50 values were calculated using the Graphpad Prism software. GI50 was defined as the concentration that led to 50% viable cells.
5
MDA-MB-231 Cell Proliferation Assay
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MDA-MB-231 cells were harvested and seeded at a density of 1000 cells/well in a 96 well plate. Cell proliferation was quantified using the CellTiter 96 Aqueous Non-Radioactive Proliferation Assay (Promega). Briefly, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carbomethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosulfate (PMS) were combined and added to phenol red free RPMI media at a 1 to 5 dilution. The MTS solution was added to wells at daily time points for 4 days. After incubation for 4 hours, formazan levels were quantified using a Cytation3 plate reader (BioTek) at an absorbance wavelength of 490 nm. All formazan readings were normalized to the standard curve and to the initial time point. Statistical significance at each time point was determined using two-way ANOVA with p < 0.05. Error bars on all proliferation data represent the standard error of the sample mean.
6
Cytotoxicity Assay of Metal Complexes
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Normal dermal fibroblasts and cancer cell lines HCT116 and A2780 were seeded in 96-well plates with a density of 7500 cells per well. After 24 h, culture media was replaced, and cells were exposed to different concentrations of complexes 1 –3 or DMSO 0.1% (v/v) (vehicle control) or cisplatin (positive control) for 48 h (Figure S14 ). Cells exposed to cisplatin were used as a positive control [103 (link),104 (link)]. After 48 h of incubation, the CellTiter 96® Aqueous Non-Radioactive Proliferation assay (Promega, Madison, WI, USA) was used to determine cellular viability through the production of formazan through the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) by dehydrogenases present in metabolically active cells [103 (link),104 (link)]. The amount of formazan can be determined by its absorbance at 492 nm in an Infinite M200 microplate reader (Tecan, Mannedorf, Switzerland) [103 (link),104 (link)]. The biological activity of the complexes was compared using the half maximal inhibitory concentration of cellular proliferation (IC50) determined with Prism 8.2.1 software for windows (GraphPad Software, La Jolla, CA, USA).
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