Feb 27, 2023
MTT (Assay protocol
- Abdulkareem Hameed Abd1,
- Enas Jawad Kadhim2,
- Matin Mahmood3
- 1Department of Pharmacology, College of Medicine, Al-Nahrain University, Kadhimiya, Baghdad, Iraq;
- 23 Department of Pharmacognosy and Medicinal Plants, College of Pharmacy, University of Baghdad, Baghdad, Iraq;
- 3Department of Pharmacology, College of Pharmacy, Alkitab University, Altun Kopre, Kirkuk, Iraq
Protocol Citation: Abdulkareem Hameed Abd, Enas Jawad Kadhim, Matin Mahmood 2023. MTT (Assay protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly72emlx9/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 22, 2023
Last Modified: February 27, 2023
Protocol Integer ID: 77471
Abstract
MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)) is used to assess cell viability as a function of redox potential. Actively respiring cells convert the water-soluble MTT to an insoluble purple formazan.
AMJ13: 2 (Ahmed.Murtuda ,Jabria 2013)cell line:
The cell line of breast cancer has been obtained from Iraqi breast cancer which originated from the prime tumor of an old Iraqi woman (70 years) with a histological identification with carcinoma of infiltrating ductal (1).
SK-GT-4:esophageal carcinomacell line was established from a primary tumors in 1989 from a 89 year-old Caucasian male who presented with dysphagia secondary to a well-differentiated adenocarcinoma arising in the Barrett epithelium of the distal oesophagus
Materials
| No. | Item | Company | Country | |
| 1 | incubator | Cypress Diagnostics | Belgium | |
| 2 | Microtiter reader | Gennex Lab | USA | |
| 3 | Laminar flow hood | K & K Scientific Supplier | Korea | |
| 4 | Micropipette | Cypress Diagnostics | Belgium | |
| 5 | Cell culture plates | Santa Cruz Biotechnology | USA |
| No. | Items | Company | Country | |
| 1 | Trypsin/EDTA | Capricorn | Germany | |
| 2 | DMSO | Santacruz Biotechnology | USA | |
| 3 | RPMI 1640 | Capricorn | Germany | |
| 4 | MTT stain | Bio-World | USA | |
| 5 | Fetal bovine serum | Capricorn | Germany |
Maintenance of Cell lines
Maintenance of Cell lines
SK-GT-4 cell line, was maintained in MEM supplemented with 10% Fetal bovine, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cells were passaged using Trypsin-EDTA reseeded at 50% confluence twice a week, and incubated at 37 °C
NHF cell line, was maintained in MEM supplemented with 10% Fetal bovine, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cells were passaged using Trypsin-EDTA reseeded at 50% confluence twice a week, and incubated at 37 °C
MTT Assay
MTT Assay
To determine the cytotoxic effect, the MTT cell viability assay was conducted on 96-well plates
Cell lines were seeded at 1 × 104cells/well. After 24 hrs. or a confluent monolayer was achieved, cells were treated with tested compound
Cell viability was measured after 72h of treatment by removing the medium
adding 28 µL of 2 mg/mL solution of MTT
incubating the cells for 1.5 h at 37 °C.
After removing the MTT solution, the crystals remaining in the wells were solubilized by the addition of 130 µL of DMSO (Dimethyl Sulphoxide)
followed by 37 °C incubation for 15 min with shaking (orbital shaker)
The absorbency was determined on a microplate reader at 492 nm (test wavelength)
The assay was performed in triplicate
The inhibition rate of cell growth (the percentage of cytotoxicity) was calculated as the following equation
Protocol references
- Al-Shammari, A. M., Alshami, M. A., Umran, M. A., Almukhtar, A. A., Yaseen, N. Y., Raad, K., & Hussien, A. A. (2015). Establishment and characterization of a receptor-negative, hormone-nonresponsive breast cancer cell line from an Iraqi patient. Breast Cancer: Targets and Therapy, 7, 223.
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- Al-Shammari AM, Alshami MA, Umran MA, Almukhtar AA, Yaseen NY, Raad K, et al. Establishment and characterization of a receptor-negative, hormone-nonresponsive breast cancer cell line from an Iraqi patient. Breast Cancer: Targets Ther. 2015;7:223-30.
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- Al-Ziaydi AG, Al-Shammari AM, Hamzah MI, Kadhim HS, Jabir MS. Newcastle disease virus suppress glycolysis pathway and induce breast cancer cells death. VirusDisease. 2020;31(3):341-8.