For clonogenic assays, cells were treated with compound for 48 h, media were removed and replaced with fresh media for a further 12 days to measure colony formation. For cell viability assays, lymphoma cells were plated at 20,000 cells in 200 μl per well in U-bottom 96 well plates in RPMI medium +20 % FCS. Cells were re-suspended 48 h later, diluted 10-fold in PBS + propidium iodide (PI) and the concentration of PI negative cells was counted using an Attune flow cytometer with autosampler. Breast cancer cells were seeded at a density of 10,000 cells/well in a sterile 96 clear-well plate with 150 μl of DMEM (+10 % FCS and 2 mM L-Glutamine). Each compound treatment was performed in triplicate for 72 h at concentrations of 100 nM and 1, 5, 10 and 50 μM in 100 μl of full-medium. After 72 h, 20 μl of MTT solution (3 mg of MTT Formazan, Sigma/1 ml PBS) was added to the medium and incubated for 4 h at 37 °C in a CO2 incubator. The MTT-product was solubilised with 100 μl DMSO, and for 1 h, incubated in the dark at room temperature. The optical density was read at 570 nm with PHERAstar.
Mtt formazan
Manufactured by Merck Group
Sourced in United States
MTT Formazan is a colorimetric assay used for measuring the activity of enzymes that reduce the yellow tetrazolium dye MTT to its insoluble purple formazan product. This reaction takes place in the mitochondria of living cells, making it a useful tool for assessing cell viability and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Trypsinase, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Penicillin g, by Thermo Fisher Scientific (2 mentions)
Spectrostar nano microplate reader, by BMG Labtech (1 mentions)
Dulbecco s phosphate buffer saline, by Thermo Fisher Scientific (1 mentions)
Versamax elisa microplate reader, by Molecular Devices (1 mentions)
Thiazolyl blue formazan, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Antibiotic antimycotic 100x, by Thermo Fisher Scientific (1 mentions)
Annexin 5 apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Elisa plate reader, by Agilent Technologies (1 mentions)
Dmem f12, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Chloroform, by Merck Group (1 mentions)
Dimethylformamide, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Multiskan go, by Thermo Fisher Scientific (1 mentions)
Cytotoxicity detection kitplus ldh, by Roche (1 mentions)
14 protocols using mtt formazan
1
Cytotoxicity and Colony Formation Assays
2
Evaluating Glioblastoma Cell Viability
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To assess cell viability, 96-well plates were coated with Matrigel under ice-cold conditions prior to plating with cells. Adherent glioblastoma cell lines were passaged and seeded at 4000 Cells/well in 96-well plates overnight, before treatment with a clinically relevant dose of TMZ (35 µM) [14 (link)] or RT (2 Gy). After 7 days, 50 µL of a 2 mg/mL solution of MTT Formazan (Sigma-Aldrich, USA) and Dulbecco’s Phosphate Buffer Saline (DPBS, without magnesium chloride and calcium chloride GibcoTM, Waltham, MA, USA) was added to each well and incubated for 3 h. The medium/MTT solution was aspirated and DMSO (120 µL) added into each well. Each plate was shaken using an IKA® MS 3 basic shaker (Sigma Aldrich, Saint Louis, MO, USA) at 600 rpm for 2 min. Absorbance was read at 570 nM using the SPECTROstar®Nano microplate reader (BMG LABTECH, Ortenberg, Germany).
3
Cytotoxicity of HIV201B2 and U78Ab3
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SH-SY5Y cells cultured in DMEM medium were treated with HIV201B2 and U78Ab3 with increasing concentrations (2, 10, 50, and 250 µg/mL) for 24 h. Cell viability was measured using the MTT formazan (Sigma Aldrich, St. Louis, MO, USA) assays following the manufacturer’s instructions and reading the absorbance at 570 nm using a standard spectrophotometer.
4
Saos-2 Cell Viability Assay
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To assess the cell viability rate, the Saos-2 were exposed to the experimental material extracts for the periods of 1, 3, and 7 days. The cells exposed to the culture medium were the positive control of viability (CT). After the incubation periods, the medium was changed to 900 µL of DMEM medium without FBS and 100 µL of MTT solution (5 mg/mL of MTT Formazan, Sigma-Aldrich, St. Louis, MO, USA). The culture plates were incubated for an additional 4 h at 37°C, 5% of CO2, and 95% humidity. After that, the MTT solution was washed and the crystals formed at the bottom of the culture plates were solubilized in 500 µL acid isopropyl alcohol (HCl; isopropyl alcohol, 0.04N). Samples containing 100 µL were transferred to 96-well plates (TPP - Techno Plastic Products, Zollstrasse, Trasadingen, Switzerland) and analyzed in an automatic microplate reader (VersaMax ELISA Microplate Reader, Molecular Devices, Sunnyvale, CA, USA) set to an optical density of 570 nm22. The experiment was repeated three times independently and the obtained data were analyzed by ANOVA and Tukey’s-HSD post-hoc test (level of significance: p≤0.05).
5
In Vitro Culture of Fish Brain Cells
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MTT Formazan (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan, thiazolyl blue formazan (CAS Number: 57360-69-7) and bovine serum albumin (BSA) were purchased from Sigma Chemical (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM), L-15 medium (Leibovitz), antibiotic–antimycotic (100X) and fetal bovine serum (FBS) were purchased from GIBCO BRL (Grand Island, NY, USA). Primary fish cell cultures were obtained from the cell and tissue culture laboratory of the Centro de Investigación en Alimentación y Desarrollo, A. C. Mazatlán (Center for Research in Food and Development). All procedures and protocols were performed under the Official Mexican Standard (NOM-033-ZOO-1995 regulation, which refers to the humanitarian sacrifice of domestic and wild animals); the protocols are in accordance with international guidelines for the use of animals in research.
Cell cultures of C. viridis brain cells were maintained at 27 °C in a humidified 5% CO2 atmosphere with growth medium (DMEM medium containing 10% fetal bovine serum, 1% of penicillin + streptomycin, 100 U/mL of penicillin + 100 µg/mL of streptomycin) changed every other day.
Cell cultures of C. viridis brain cells were maintained at 27 °C in a humidified 5% CO2 atmosphere with growth medium (DMEM medium containing 10% fetal bovine serum, 1% of penicillin + streptomycin, 100 U/mL of penicillin + 100 µg/mL of streptomycin) changed every other day.
6
MTT Cell Viability Assay Protocol
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Drug cytotoxicity and dose–response curves were performed using a standard MTT cell viability assay [37 (link)]. Primary VSMC were plated at 7000 cells/well in a 96-well plate. Drug incubation was established for 2–24 h incubation (37 °C with 95% oxygen and 5% CO2) with a serial drug dilution in DMEM. After each drug incubation period, a further 6 h incubation with 50 μL of 5 mg/mL MTT Formazan (Sigma Aldrich 57360-69-7) was added to each well, wrapped in foil, and incubated in the same way. At the end of the MTT incubation period, all media was replaced with 100 μL of 99.5% dimethyl sulphoxide (DMSO) and 50 μL of glycine buffer (0.1 M glycine and 0.1 M NaCl adjusted to 10.5 pH with 1 M NaOH). The plate’s absorbance was immediately read at 560 nm using a VictorTM X3 PerkinElmer plate reader (PerkinElmer, Waltham, MA, USA).
7
Evaluating GCSC Viability and Apoptosis
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After GCSCs were transfected for 24, 48, and 72 h, 1-(4, 5-dimethylthiazol-2-yl)-3, 5-diphenylformazan thiazolyl blue formazan (MTT formazan; Sigma, St. Louis, MO, USA) was added to the wells and incubated at 37 °C for 5 h. Then, 150 μl DMSO was added to each well; the 96-well plates were rotated for 10 min to dissolve the purple crystals, and the absorbance at 490 nm was measured using an ELISA plate reader (Biotek Instruments, Highland Park, VT, USA). Apoptosis and the cell cycle were analyzed in GCSCs transfected for 48 h using the Annexin V Apoptosis Detection Kit (eBioscience, San Diego, CA, USA) and ribonuclease (Worthington Biochemical Corp, Lakewood, NJ, USA) according to the manufacturer’s instructions using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
8
P3HB/CUR/MCNT Composite Fabrication
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P3HB powder with the formulation of –[COCH2CH(CH3)O]n - and CAS Number: 29435-48-1, CUR (CUR)(CAS Number: 458-37-7), chloroform (formula: CHCl3, CAS Number: 67-66-3), dimethylformamide (DMF) (formula: HCON(CH3)2, CAS Number: 68-12-2), dimethyl sulfoxide (DMSO) (Formula: (CH3)2SO, CAS Number: 67-68-5), DMEM F12, fetal bovine serum (FBS) (MDL Number: MFCD00132239) and MTT formazan (CAS Number: 57360-69-7) were purchased from Sigma-Aldrich-Merck (St. Louis, MO, USA). MCNTs (5‒25 nm in diameter, 0.5‒2 μm in length, and >95wt%. of purity) functionalized with carboxylic groups (–COOH) were obtained from US Research Nanomaterials (Houston, TX, USA).
9
MTT Cytotoxicity Assay Protocol
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3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), MTT formazan, Triton X-100, etylenediaminetetraacetic acid (EDTA) were obtained from Sigma-Aldrich, Poland. Organic solvents came from POCh, Poland. All chemicals were of analytical purity grade and did not undergo further purifications.
10
Cell Viability Assay of hBMSCs
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MTT assay was used to determine the cell viability of hBMSCs following manufacturer’s instruction. Cells were seeded in 96-well plate at 1×104/well density for 24 h, incubated with HAND2 nanoparticles in different concentrations. After 72 h, assays were performed by adding 50 μL of MTT solution (5 mg/mL MTT formazan, Sigma-Aldrich). The culture plate then incubated for an additional 4 h at 37°C, 5% CO2, and 95% humidity. After removal of MTT solution, the crystals formed at the bottom of the culture plate were solubilized in 150 μL of acid isopropyl alcohol, recording the absorbance at 570 nm with an automatic microplate reader (Multiskan GO, Thermo Fisher, USA). The quantity of formazan product as measured is directly proportional to the cell viability. The experiment was repeated by three times.
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