Cell viability was assessed by the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Cells were treated with increasing drug concentrations for 72 h (TC32, EW8, Rh30, and RD), 120 h (JN-DSRCT-1 and Rh41), or 144 h (Rh18), based on the estimated cell division rate. MTS solution (CellTiter 96 Aqueous Solution Cell Proliferation Assay, Promega, WI, USA) was added (10 μl), and plates were incubated for 2 h at 37°C. Extinction was measured at 490 nm (iMark microplate absorbance reader, Bio-Rad, CA, USA). IC50 values were calculated with GraphPad Prism version 5.03 software.
Celltiter 96 aqueous solution cell proliferation assay
Manufactured by Promega
Sourced in United States
The CellTiter 96 Aqueous Solution Cell Proliferation Assay is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays. The assay quantifies the amount of a formazan product that is directly proportional to the number of living cells in culture.
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Lab products found in correlation
Fitc annexin 5 apoptosis detection kit, by BD (4 mentions)
Pi rnase staining buffer, by BD (3 mentions)
Penicillin, by Merck Group (3 mentions)
Streptomycin, by Merck Group (3 mentions)
Phosphate buffered saline (pbs), by Corning (3 mentions)
Synergy ht, by Agilent Technologies (3 mentions)
Imark microplate absorbance reader, by Bio-Rad (2 mentions)
Trypsin, by Corning (2 mentions)
Mccoy s 5a, by Corning (2 mentions)
Glutamine, by Corning (1 mentions)
Mccoy s 5a medium, by Corning (1 mentions)
Wogonin, by Merck Group (1 mentions)
Baicalein, by Merck Group (1 mentions)
Rpmi 1640, by Corning (1 mentions)
Leibovitz s l 15, by Corning (1 mentions)
C0775, by Merck Group (1 mentions)
T8154, by Merck Group (1 mentions)
F8775, by Merck Group (1 mentions)
Glomax multi detection system, by Promega (1 mentions)
Formic acid of hplc grade, by Merck Group (1 mentions)
13 protocols using celltiter 96 aqueous solution cell proliferation assay
1
Cell Viability Assessment by MTS Assay
2
Cell Proliferation and Apoptosis Assay
3
MTS Assay for 3D Scaffold Viability
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Quantitative assessment of cell viability/proliferation in the scaffolds took place by measuring the increase of metabolically active cells using the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] MTS reagent (Promega, CellTiter96® Aqueous Solution Cell Proliferation Assay, WI USA). The reagent was supplemented to fresh culture medium at a ratio of 1 : 5 according to the manufacturer's protocol and added to the 3D scaffold cultures prior to a 3 h incubation at 37 °C, according to the manufacturer's protocol. Afterwards, the absorbance was measured at 490 nm on a plate reader (Synergy HT, BioTek, VT, USA).
4
Assessing Cell Viability and Migration
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Cell viability was assessed by MTS assays. All cells were seeded at 5000 cells per 100 μl/well. Cells were allowed to adhere and treated with varying drug concentrations for 120 h, based on the estimated growth rate of JN-DSRCT-1 cells. MTS solution (CellTiter 96 Aqueous Solution Cell Proliferation Assay, Promega, WI, USA) was added (10 µl) and plates were incubated for 2 h at 37 °C. Extinction was measured at 490 nm (iMark Microplate Absorbance Reader, Bio-Rad, CA, USA). IC50 values were calculated using GraphPad Prism Version 5.03 software.
Effects of treatment on cell migration were assessed by wound healing assays as previously described (van Erp et al. 2017 (link)). Cell migration is depicted in relative gap size: gap size at tN/gap size at t0 (tN = hours of treatment, t0 = start of treatment). Differences in gap size were analyzed by 2-way ANOVA with Bonferroni posttest, p value < 0.05 was considered significant (*< 0.05, **< 0.01, ***< 0.001).
Effects of treatment on cell migration were assessed by wound healing assays as previously described (van Erp et al. 2017 (link)). Cell migration is depicted in relative gap size: gap size at tN/gap size at t0 (tN = hours of treatment, t0 = start of treatment). Differences in gap size were analyzed by 2-way ANOVA with Bonferroni posttest, p value < 0.05 was considered significant (*< 0.05, **< 0.01, ***< 0.001).
5
Evaluating Baicalin and Wogonin Cytotoxicity
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All cell culture plasticware were obtained from Falcon Labware (Franklin Lakes, NJ, USA) and Techno Plastic Products (Trasadingen, Switzerland). Trypsin, McCoy's 5A, Leibovitz's L-15, RPMI-1640 and DMEM media, and phosphate-buffered saline were obtained from Mediatech, Inc. (Herndon, VA, USA). Penicillin and streptomycin were obtained from Sigma-Aldrich (St. Louis, MO, USA). The MTS assay kit, CellTiter 96 Aqueous Solution Cell Proliferation Assay, was obtained from Promega (Madison, WI, USA). The Annexin V-FITC apoptosis detection kit was obtained from BD Biosciences (Rockville, MD, USA). PI/RNase staining buffer was obtained from BD Biosciences Pharmingen (San Diego, CA, USA). Caspase 3 and 9 ELISA kits were obtained from BioVison (Mountain View, CA, USA). Baicalin and wogonoside were obtained from Indofine Chemical Co. Inc. (Hillsborough Township, NJ, USA). Baicalein and wogonin were obtained from Sigma-Aldrich.
6
Effect of Medications on Fibroblast Proliferation
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To study the effect of different medication on the proliferation of Fib, the cells were cultured in the expansion medium described above supplemented with 10 nM or 1 μM dexamethasone, 0.05 mg/ml or 0.5 mg/ml diclofenac, or 100 nM or 200 nM recombinant human decorin. Fib cultured with standard expansion medium served as controls. Fib was seeded in 96-well plates at density of 5 × 104 cells/well. After one, three, five, and seven days, the proliferation of Fib was determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS; Cell Titer 96 Aqueous Solution Cell Proliferation Assay, Promega, Madison, Wisconsin) following the manufacturer’s instructions. Briefly, culture medium was removed and the cells were incubated with 100 µl MTS, the concentration of which was 0.5 mg/ml. After two hours of incubation at 37 °C in 5% CO2, optical density (OD) was determined at 490 nm using a 96-well plate reader.
7
Wogonoside and Wogonin Cytotoxicity Assay
8
Metabolic Activity and Proliferation of hASCs on Magnesium Scaffolds
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Metabolic activity of hASCs cultured on magSPCL scaffolds was evaluated by MTS assay (Cell Titer 96 Aqueous Solution Cell Proliferation Assay, Promega). Briefly, after 7, 14 and 21 days of culture, the constructs were transferred to another multiwell plate, washed with PBS and incubated for 3 h at 37 °C and 5% CO 2 atmosphere in a mixture of serum-free culture medium without phenol red and MTS solution (5:1 ratio). Then, 100 μL of each sample was transferred to a 96 well-plate and the absorbance read at 490 nm (Synergy HT, Bio-TeK Instruments). The samples were read in triplicate and a blank reading was also performed for correction purposes. The proliferation of hASCs was determined through DNA quantification of cell lysates, using the Quant-iT TM PicoGreen R dsDNA kit (P7589, ThermoFisher), after 7, 14 and 21 days of culture. Samples were collected into microtubes with 1 mL of ultrapure water and stored at -80 °C. Then, samples were thawed, sonicated, and the supernatant analysed using a microplate reader with an excitation of 485/20 nm and an emission of 528/20 nm (Synergy HT, Bio-TeK Instruments). Samples and standards were made in triplicate.
9
Metformin-Induced Cell Viability Assays
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Cell viability after prolonged metformin treatment was assessed with different protocols. In the clonogenicity assay cells (2 × 103) were plated in 100 mm dishes to allow clones formation. At the end of metformin incubation, plates were washed twice with a phosphate buffered saline solution (PBS; 79382; Sigma-Aldrich) and fixed with 4% formaldehyde solution in PBS (F8775; Sigma-Aldrich) at room temperature (rt). After 10 min, dishes were washed twice in PBS and stained for 5 min with 0.5% crystal violet (C0775; Sigma-Aldrich). Finally, cells were washed with distilled water and air-dried. The colonies were counted the following day. In trypan blue exclusion assay, cells were seeded on 6-well plates. Following treatments, cells were harvested and stained with 0.4% trypan blue (T8154; Sigma-Aldrich). The cell suspension was applied to a haemocytometer and counted with a phase contrast microscopy (NIKON EclipseTE2000U, Nikon Netherlands, Amsterdam, The Netherlands). Finally, cell viability was checked also by CellTiter96® AQueous Solution Cell Proliferation Assay (G3580; Promega). Cells were seeded in 96-well plates. Following metformin treatments, 20 µL of CellTiter 96® Aqueous Solution was added to 100 µL of culture medium. After 2 h of incubation at 37 °C, absorbance at 490 nm was measured with the GloMax®-Multi Detection System (Promega).
10
HPLC-Based Phytochemical Profiling and Bioactivity Evaluation
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Reference compounds including ginsenoside Rb1 and compound K were obtained from Indofine Chemical Company (Somerville, NJ, USA) and ChromaDex Inc. (Irvine, CA, USA), respectively, whose purities were more than 98% determined by HPLC-DAD. General anaerobic medium (GAM) was obtained from Kayon Biological Technology Co. Ltd. (Shanghai, China). Acetonitrile (ACN) and formic acid of HPLC grade were from Merck KGaA (Darmstadt, Germany). Deionized water (18 MΩ-cm) was supplied with a Milli-Q water system (Millipore, Milford, MA, USA). Trypsin, McCoy's 5A, and phosphate-buffered saline were obtained from Mediatech, Inc. (Herndon, VA, USA). Penicillin and streptomycin were obtained from Sigma-Aldrich (St. Louis, MO, USA). An MTS assay kit, CellTiter 96 Aqueous Solution Cell Proliferation Assay, was obtained from Promega (Madison, WI, USA). A FITC-Annexin V apoptosis detection kit was obtained from BD Biosciences (Rockville, MD, USA). PI/RNase staining buffer was obtained from BD Biosciences Pharmingen (San Diego, CA, USA). All cell culture plasticware were obtained from Falcon Labware (Franklin Lakes, NJ, USA) and Techno Plastic Products (Trasadingen, Switzerland).
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