Use the 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT, Sigma-Aldrich) as described by Mosman (1983, PMID ). Briefly, cells were plated in 96-well plates and treated accordingly. After treatment, 5µl MTT in PBS (5mg/ml) was added per 100µl culture medium and cells were incubated for 2h at 37°C. Next, the medium was discarded and 100µl isopropanol with 0.04N HCl was added to the cells. Plates were shaken for 30 minutes and absorptions were measured at 570nm using a Multiskan FC plate reader (Thermo Fischer). All measurements were performed in triplicates.
Roche sells a very nice "ready to use" kit for that. It is based on the same method that Bjorn described, but uses detergent instead of HCl to lyse the cells.
http://www.roche-applied-science.com/proddata/gpip/3_5_3_21_1_5.html
I have used MTT before but was badly criticized by a reviewer about its use for proliferation assay. The assay relies upon mitochondrial function so if the treatment also affect mitochondral function the interpretation needs to be careful.
Ru-Jeng is rigth on that the method is based on mitochondrial function. Actually is measuring the amount of cells alive (with functional mitochondria), and not directly cell proliferation. But the idea is that the number of cells increases if the cells proliferate. The nice thing is that if the cells die somehow they do not give a positive signal since their mitochondria are not intact.
You should take care that whatever treatment you do to your cells do not affect mitochondria function (you can actually test measuring on a known number of cells treated or not whether the MTT signal is the same).
Method: Grow cells in required medium (PH 7.4) supplemented with
10% FBS and seed at a cell density of 10,000cells/well in a 96 well plate and incubated overnight at 37C in CO2 incubator and the next day add any drug you are testing and incubate at 370C for 48hrs.
After 48hrs into each well add 10ul of MTT (4mg/ml) and incubate for 3hrs at 37C in CO2 incubator and then decant the plate; dissolve the formazan crystals in 100ul of DMSO per well and leave on gentle shaking for 10mts. Read the plate at 570nm in an ELISA reader.
Calculation : % inhibition of Growth = 100-(( Test CPM/ Control CPM)*100).
hope this helps.
- Badges
- Science topic
- Similar topics
- Cell Biology
- Methods