My colleague tried to measure cell survival using MTT assay, but he didn't get any signal
My colleague running experiment to test the effect of some drugs on some cancer cell lines. He used the MTT assay to measure the survival in 96 well plate. He used MSC from human BM as a control. The effect was dose dependent on cell lines but he couldn’t detect the signal on MSC even their morphology look fine and they grow very well.
Actually it is almost the same protocol that i use:
1. Seed the cells 100 µl (104 per well) in 96 well plate.
2. Incubate at 37oC for 24 hours.
3. Add 100 µl of 5 mg/ml MTT stock solution to each well.
4. Incubate at 37 C for 4 hours.
5. Carefully remove the media and add 100 µl of SDS 10% to each well.
6. Cover the plate with saran paper
7. Incubate at 37 C over night.
But as Dr. Souchelnytskyi said, the type of cells and the number of seeded cells in a crucial factor in the MTT.
Also when you remove the and you add the SDS, try to avoid removing the cells, which is a common mistake which will cause the absent of the color
What is the exact protocol he used? the MTT dose? the solubilizing agent for the formazon formed ? the incubation time
Many thanks Dr. Yacoub, I sent your reply to my colleague and either he will communicate with you directly or I will get you his answer.
Questions of Dr Yacoub are very relevant. It is important to exclude any technical problems. There are many points to be controlled for these technical points, such as state of the cells, number of cells, concentration of reagents, type of media you use, temperature, incubation time, you have to have positive and negative controls, quality of reagents, etc. Then, there are also issues related to the cells you test.
Your comment indicate that it may be features of MSC cells which would require optimization of MTT assay, as MTT worked with other cells.
In such situations, I ask my guys to run optimization - times, concentrations and reading are the first to check.
Good luck!
This is the MTT condition that Dr. Tamimi used:
the MTT dose? = 10ul of (5mg/ml MTT product) in 100 ul of the media used.
the solubilizing agent for the formazan formed ? = 0.1g SDS in 1 ml of 0.01M HCl
incubation time = 4 hours at 37C for MTT essay
from 4 to 18 hour (overnight incubation) at 37 C for solubilizing reagent
Actually it is almost the same protocol that i use:
1. Seed the cells 100 µl (104 per well) in 96 well plate.
2. Incubate at 37oC for 24 hours.
3. Add 100 µl of 5 mg/ml MTT stock solution to each well.
4. Incubate at 37 C for 4 hours.
5. Carefully remove the media and add 100 µl of SDS 10% to each well.
6. Cover the plate with saran paper
7. Incubate at 37 C over night.
But as Dr. Souchelnytskyi said, the type of cells and the number of seeded cells in a crucial factor in the MTT.
Also when you remove the and you add the SDS, try to avoid removing the cells, which is a common mistake which will cause the absent of the color
Many thanks Dr. Yacoub for the excellent feedback and going through the protocol.
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