Thiazolyl Blue Tetrazolium Bromide

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- 250 μg/ml of MTT is added and incubated for 4 extra hours	- Absorbance is measured at a wavelength of 570 nm with background subtraction at 630-690 nm.

Protocol tips
- 250 μg/ml of MTT is added and incubated for 4 extra hours
Downstream tips
- Absorbance is measured at a wavelength of 570 nm with background subtraction at 630-690 nm.

Publication protocol

HeLa human cervical cancer cells were from the American Type Culture Collection and were maintained in DMEM medium (Mediatech, Inc.) containing 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 μg/ml). HeLa cells were transfected with Safectine RU50 (SydLabs, Natick, MA) according to the manufacturer's protocol. For cell cycle analysis, cells were washed with phosphate buffered saline (PBS) and fixed with 70% ethanol. After centrifugation, cells were washed and resuspended in PBS containing 0.25 mg/ml propidium iodide (PI) and 10 mg/ml RNase A (Sigma, St. Louis, MO, USA). We used MTT assay to measure the cell proliferation. MTT [Thiazolyl Blue Tetrazolium Bromide] (CAS Number: 298-93-1) was purchased from RPI corp (Mt. Prospect, Il). Cells were suspended in DMEM containing 10% FBS, seeded in 96-well plates, and then incubated for an overnight. Next day, wells’ medium was replaced with 100 μL of FBS free medium containing 250 μg/ml MTT and incubated for 4 extra hours. Then supernatants were removed and 200 μL DMSO was added to each well. Plates were shaken for 10 min and the absorbance was measured at 570 nm by Cytation 5 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT)

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Manufacturer protocol

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