I need to pre-treat my cells with a plant extract first. What is the recommended incubation time for my cells to be exposed to my extract for this MTT assay- 24 hours or 48 hours? I'm using fibroblast cells SW872.
I suggest to treat for both 24 and 48 hrs for couple of times and then depending upon the cell viability or your extract toxicity you can determine whether you need 24 or 48 hrs of treatment.
This depends on the plant extract doses that are used in the test. To decide for an effective dose for a distinct time point it is important to check the behavior of the cells first.
You should check the cell viability by a light microscope and to which extend cells change their morphology or become detached from the cell culture plate. Use your eyes! If you dont see effects after 24 h, then check the cells later at 48h. If there is a clear dose-dependency you should perform the MTT assay at this time point.
I suggest, as Gajendra Sherestha, to treat the cells both for 24 and 48 h. However, be careful to the cell density in the culture plate used. It also reccomended for MTT- assay to hormone deprive the cells before the test.
I suggest, 20 h exposure to the extract, followed by adding of MTT for 4h. Perform the measurement of Abs within 15 to 20 min after 24'th h. Mark that your following experiments with the chosen extract concentration/s should not exceed 24 h. According the treatment time you should choose the exposure time for your MTT test.
We do it like that and it works perfectly!
Good luck!
Hi Jhoti,
Cells (1 × 105 cells/mL) were plated in 96-well microtiter plates and incubated overnight for cell attachment. The medium was then removed and fresh medium, along with the corresponding test concentrations (0.001–1000 μg/mL) were added to the cultures. Following treatment for 24–72 h at 37 °C, MTT solution (5 mg/mL in PBS) was added and incubation was prolonged for 4 h at 37 °C. Inhibition of cell proliferation was monitored by MTT (Mishra et al., 2014).
You can do dose and time dependent studies with your plant extract. Treat cells for 24 h, 48 h and 72 h , followed by adding of MTT for 4h and measure absorbance after 15 min. Simultaneously, you can observe cells under phase contrast microscope for morphometric changes occurring after treatment with plant extract (24 -72 h).
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