I have conducted 2 MTT assays on a herbal product at different time points.
The first time, I seeded my cells for 24h, treated the cells for 24h and added MTT solution (0.5 mg/ml) for 72 hours. The readings were between 0.6 - 0.8 at 570nm.
The second time, I changed my treatment time to 72 hours but reduced my MTT incubation time to 4 hours.
Surprisingly, I was not able to get any results in the second run. When I removed the MTT solution, I saw a lot of the crystals were being sucked out too.
Could there be a problem in my pipetting skills or my protocol? Please help.
Dear Abbirami Balachandran unfortunately I'm not a specialist in this field enough to give you a qualified answer to your interesting technical question. However, you might want to have a look at the answers given to the following closely related questions which have been asked earlier on RG:
Can I use MTT protocol for short time incubation?
https://www.researchgate.net/post/Can_I_use_MTT_protocol_tfor_short_time_incubation
(14 answers)
and
A doubt about MTT assay
https://www.researchgate.net/post/A_doubt_about_MTT_assay2
(12 answers)
In any case it's interesting to see that other researchers faced similar problems in the past. Good luck with your research and best wishes!
Hello Abbirami Balachandran
It won't be proper to treat cells with MTT for 72 hours. The conversion of MTT to formazan crystals by cells in culture is time dependent. Longer incubation time will result in accumulation of color and increased sensitivity up to a point. However, the incubation time with MTT is limited because of the cytotoxic nature of the detection reagent which utilize energy such as reducing equivalents like NADH from cells to generate a signal.
Culture conditions that alter the metabolism of cells will likely affect the rate of MTT reduction into formazan. So, the incubation time for MTT in MTT assay is from a minimum of 30 mins to a maximum of 4 hours depending on the cell type and culture conditions. You can extend MTT incubation time to 6 hours in certain cases.
You could modify your protocol. Treat your cells with the herbal product at different time points such as 24, 48 and 72 hours keeping the MTT incubation time constant at 4 hours for all the time points.
Hope this helps.
Good Luck.
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