MTT assay concentration fixing stock solution perpetration and require volume for test.
This is a part of the methodology I am using for my research:
"...exponentially growing lymphoma L5178Y-R cells were plated at 5 X 104 cells/mL in flat-bottomed 96-well plates in 100 µL of complete RPMI 1640 medium. These tumor cell cultures were then incubated for 44 h at 37 oC in 5% CO2 in the presence of starting from 1mg/mL and decresasing, of the extracts in a volume of 100 µL (the extracts were dissolved in 10 % ethanol as explained previously, and vehicles were tested using the same concentration of the solvent but without the extract; vincristine was used as positive control). After incubation, 15 µL of MTT (0.5 mg/mL) were added to all wells, and cultures were additionally incubated for 4 h. Next, the plates were decantated and added 80 µL of DMSO to each well. Optical densities were then read in a microplate reader at 570 nm."
For any other question, I am at your service.
Hi there,
It really depends on the solvents used in the plant extracts. But usually for adhesion cells, you need to plate out the cells first and let them adhere and incubator for 4 to 6 hours before adding your extracts.
You will need to do colour control should your extract interfere with the colour of the medium used.
I am not 100% sure about the starting concentration (screening concentration), so when I have the information I will post it.
As for starting volume, subject to the compound availability, I use 20 uL drug and 180 uL cell lysate adjusted to whatever suitable cell number for your assay.
With the MTT, we read at the test wavelength of 540 nm and a reference wavelength at 690 nm (this is based on the Mossman paper).
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