Hi everyone,
I'm doing MTT assay to test the cytotoxicity of 4 candidates on the BT-474 breast cancer cell line which rarely becomes confluent. started with 3K/well, 5K/well, 6.5K/well
left the cell two days to recover then changed the medium
but the OD of -ve control was 0.7-0.8 or less
I used doxorubicin as +ve control, the final concentration was 10MM but the % of inhibition didn't exceed 45%!!
what shall I do when the cell not confluent enough !!
You may seed higher concentration for longer time ( let's say 48 hrs) before applying treatment.
You can use a more cytotoxic agent for positive control eg. DMSO or detergents.
Try pipetting the negative control wells very gently during media change to avoid cells from uprooting from the well surface.
Hi Hamza,
What plates are you using? If this can help you, for a cell proliferation assay with CHO cells (Chinese hamster ovary) in 96-well flatbottomed plates, I seeded at 10K cells/well in 0.1 ml of medium. I think you could try the effect seeding your cells at 10-12K/well.
Your problem is that your chosen cell line rarely becomes confluent. Are you using a recommended grow medium? Even if you are they wouldn't fill the plate. Use 10000 cells per well. That should be enough for MTT to give an accurate measurement even if the cells do not proliferate in the time window of your experiment.
Another thing is that you do not measure cytotoxicity with MTT, you measure proliferation (cell number in treated and untreated wells). You can only say that you have more ore less cells then control. If you have less cells it can be from cytostatic effect of your tested compound. There are other tests for cytotoxicity to confirm cell death.
The MTT measurement required enough cell. I think that you firstly need to grow the cells in recommended media until you reach to confluence level before assess the quantification of viable cells with dye. Depending on cells, the cells density can be 7000, 10000, 50000/well.
Michele S Majoumouo Mateo Glumac María Luisa Caballero Biswajoy Ghosh
Thank you all,
This cell line is very difficult to harvest, the alive cell after harvesting a large plate not more than 5 *10^5 which not enough to seed one plate
I'll consider changing the growth medium and seeding no.
I recommend to use DMEM with 10% FBS. And seed your cells in 96 well plate 10000 cells/well. You can also increase serum level (15%) before treatment.
How the cells look like in the treated wells?
BTW cells contaminated with mycoplasma will give high background in MTT assay and may respond differently to chemotherapeutic .
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