MCF-7, MDA-MB-231, and MCF-10A cells were seeded in 96-well plates (0.8×105 cells/mL) and cultured overnight. Then, starting at 30 μg/mL, serial 1:1 dilutions of the examined compounds were added to triplicate wells on the seeded plates. The nonadherent K562 cells were loaded (0.8×105 cells/mL) on the 96-well plate, which contained the appropriate dilutions of compounds. All plates were incubated for 48 hours at 37°C, 5% CO2. After the incubation period, 20 μL of MTT (5 mg/mL) solution (Sigma-Aldrich Co) was added to all wells and incubated in darkness for 3 hours. Then, 170 μL of solution was discarded and 20 μL of dimethyl sulfoxide (DMSO) was added to each well to solubilize the purple crystals. The plates were read using a microplate reader at 570 nm (BioTek Instruments, Inc., Winooski, VT, USA). IC50 (inhibitory concentration with 50% cell viability) values, indicating the concentration of DHAQC (2) or damnacanthal that inhibited 50% of cell viability compared to untreated control, were obtained, and selective index was calculated by dividing the IC50 value of MCF-10A cells by the IC50 value of MCF-7 or MDA-MB-231 cells.
Mtt 5 mg ml solution
Manufactured by Merck Group
Sourced in Germany, United States
MTT (5 mg/mL) solution is a cell viability assay reagent. It is used to measure the metabolic activity of cells in vitro.
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5 protocols using mtt 5 mg ml solution
1
Cytotoxic Effects of DHAQC and Damnacanthal
2
Evaluating Antiproliferative Effects of Calcitriol and Tacalcitol
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Cells (1 × 105/mL) were suspended in a 10 mL culture medium and then seeded onto 96-well plates at 100 µL per well (Corning Incorporated, Corning, NY, USA). After 24 h, compounds were added in concentrations ranging from 1000 to 1 nM for 120 h. Next, 20 μL MTT (5 mg/mL) solution (Sigma–Aldrich, Steinheim, Germany) was added to each well and incubated for 4 h. After the incubation time, 80 μL of lysis buffer ((67.5 g sodium dodecyl sulfate SDS (Sigma–Aldrich, Steinheim, Germany), 225 mL N, N-Dimethylformamide (Avantor, Gliwice, Poland) and 275 mL MilliQ water IIET, Wroclaw, Poland)) were added to each well. After 24 h, a spectrophotometric measurement was carried out at 570 nm using a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). Based on the absorbance value, the percentage of proliferation inhibition and the concentration of calcitriol and tacalcitol that causes 50% inhibition of cell proliferation-IC50 (IC50, inhibitory concentration 50) was determined using the Cheburator 0.4 Dmitry Nevozhay software. A minimum of 3 independent tests were performed.
3
MTT Assay for Cell Viability
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3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to estimate the cell viability. JHU-22vect and JHU-22miR128 cells were seeded at an initial density of 5000 cells per well in a flat-bottom 96-well cell culture plate and allowed to grow for 48 hours in a humidified 5% CO2, 95% air atmosphere in an incubator maintained at 37 oC. Twenty microliters of MTT (5 mg/ml) solution (Sigma) were added to each well followed by 4-hour incubation at 37°C. After the media were removed, 200 μl of dimethyl sulfoxide were added to each well to dissolve the formazan formed. After 30 minutes incubation at room temperature, the plates were scanned with a microplate reader (Bio-Rad) that was set at 560 nm for measuring the absorbance.
4
MTT Assay for Cell Viability
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A 20 × 104 of transfected cells were seeded in a 96-wells plate, 48 h of post-transfection. Cells were incubated with 10 µl of MTT (5 mg/ml) solution (Sigma-Aldrich) and set for 2 h at 37 °C. Formazan crystals were then dissolved in DMSO, and absorbance at 570 nm was detected using a microplate reader. The average 570 nm absorbance values were used to calculate the percentage of cell viability based on the following formula: % cell viability = (OD 570 nm of sample/OD 570 nm of control) × 100.
5
Kahweol Protects INS-1 Cells from STZ-induced Cytotoxicity
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A 20 × 104 INS-1 cells were seeded in a 96-wells plate. Kahweol was purchased from Santa Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and the cells were pre-incubated with different concentrations of kahweol (2.5, 5 and 10 µM) for 24 h followed by STZ treatment (3 mM) for 3 h, as previously described [35 (link)]. Untreated cells were used as a negative control. Afterward, cells were incubated with 10 uL of MTT (5 mg/mL) solution (Sigma-Aldrich, St. Louis, MO, USA) and set for 2 h at 37 °C. Formazan crystals were then dissolved in dimethyl sulfoxide (DMSO), and absorbance at 570 nm was detected using a microplate reader. The average 570 nm absorbance values were used to calculate the percentage of cell viability based on the following formula: % cell viability = (OD 570 nm of sample/OD 570 nm of control) × 100.
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