Feb 20, 2025
- Songyot Anuchapreeda1,
- Siriporn Okonogi2,
- Mathurada Sasarom2
- 1Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai;
- 2Center of Excellence in Pharmaceutical Nanotechnology, Chiang Mai University, Chiang Mai
Protocol Citation: Songyot Anuchapreeda, Siriporn Okonogi, Mathurada Sasarom 2025. MTT assay. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbr3knlpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 20, 2025
Last Modified: February 20, 2025
Protocol Integer ID: 123057
Keywords: MTT assay, cytotoxicity, dependent cellular oxidoreductase from viable mitochondria, dependent cellular oxidoreductase, cytotoxicity of sample, mtt dye, leukemic cell line, cytotoxicity, viable mitochondria, mtt
Abstract
MTT assay was performed to investigate the cytotoxicity of samples against normal cells and leukemic cell lines according to these methods of Rueankham et al., 2023 and Moschini et al.,2023. The MTT dye is converted by NAD(P)H-dependent cellular oxidoreductase from viable mitochondria into insoluble formazan crystals.
Materials
Materials
1. PBS, pH 7.4
2. Histopaque-1077
3. RPMI-1640 containing 10% fetal calf serum, 1 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin
4. IMDM supplemented with 20% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin
5. MTT dye solution
6. DMSO
Equipment
1. A humidified incubator
2. A centrifuge
3. A microplate reader
4. A laminar flow cabinet
Troubleshooting
MTT assay
100 µL of cell suspensions at the following cell concentrations, 2.5×105 cells/mL for PBMC, 1.5×104 cells/mL for KG1a, and 1.0×104 cells/mL for K562 or Molt4 were pipetted into the well
of the flat-bottomed 96-well plates.
100 µL of each sample at concentrations ranging from 3.125 to 200 μg/mL and doxorubicin (concentrations ranging from 0.01 to 4 μg/mL) as a positive control were added to cells. A medium
without a sample was used as a negative control.
incubated for 48 h at 37°C
15 µL of 0.2 mg/mL MTT dye solution was pipetted into each well and the plate was further incubated for 4 h at 37°C.
After the supernatants of the well were removed, the formazan crystals were dissolved in 200 μL of DMSO.
The absorbance of the solutions was measured at 578 nm using 630 nm as reference wavelength.
Protocol references
1. Rueankham L, Panyajai P, Saiai A, Rungrojsakul M, Tima S, Chiampanichayakul S, et al. Biological
activities of extracts and compounds from Thai Kae-Lae (Maclura cochinchinensis (Lour.) Corner). BMC Complement Med Ther. 2023;23(1):191.
2. Moschini E, Colombo G, Chirico G, Capitani G, Dalle-Donne I, Mantecca P. Biological mechanism of cell oxidative stress and death during short-term exposure to nano CuO. Sci Rep. 2023;13(1):2326.