Hello everyone
I am trying to design an MTT experiment for an immortal adherent cell line with doubling time of 20 hours. The goal is to evaluate effects of certain growth factors and their combination on cell proliferation and determine the optimal dose.
Many papers suggested seeding 10000 cells in 96 well plates and performing MTT at 24 h intervals or at days 1, 3, 7.
I am not sure about some technical issues:
1. should I proceed with 24 h interval assay and for how many days? or the 1, 3, 7 days evaluation is good enough?
2. can I seed 5000 cells/ well in 96 well plates instead of 10000?
3. I expect my cells to become confluent after 72 hours (starting from 10000 cells), and they definitely die without medium change, so should I change the medium every 24 hours for all plates? or just change the medium after 72 hours and wouldn't this affect my results?
Thanks a lot!
Hello Shima Nazari
1. Should I proceed with 24 h interval assay and for how many days? or the 1, 3, 7 days evaluation is good enough?
You should proceed with 24 h interval assay for up to 72 hours.
2. Can I seed 5000 cells/ well in 96 well plates instead of 10000?
Since you know that cells are expected to become confluent after 72 hours (starting from 10000 cells), you should plate 7500 cells/well because plating 10000 cells/well will lead to over confluency at 72 hours and plating 5000 cells/well would be a lower seeding density for the early time point.
3. I expect my cells to become confluent after 72 hours (starting from 10000 cells), and they die without medium change, so should I change the medium every 24 hours for all plates? Or just change the medium after 72 hours and wouldn't this affect my results?
You should not change the medium every 24 hours as this will affect the results. Just leave the cells in the treatment media i.e., media containing the growth factors and their combination for 72 hours.
You may follow the protocol provided below.
1. Plate cells (7500 cells/well) in 96 well plates.
2. After 24 hours, complete medium may be exchanged with low-serum medium (media containing 1% FBS) in order to stop proliferation (cell synchronization).
3. Twenty-four hours later, the cells may be treated with media containing the growth factors and their combination. Do not forget to include negative control (cells + media + 1% FBS) and positive control (cells + media + 10% FBS), besides the test samples (cells + media + 1% FBS + growth factors and their combination).
4. Incubate the plates at 37 deg C for different time points namely, 24, 48 and 72hours.
5. After a culture period of 24, 48, and 72 hours, the plates of each time point may be removed and washed twice with sterile PBS, and the cell proliferation and viability may be evaluated using MTT assay.
Best.
Dear Malcolm
Thank you very much for you answer.
After selecting a concentration using MTT, I am going to perform my experiments in 10% serum medium and without synchronization, so, is it necessary to use reduced FBS medium in MTT assay?
Dear Shima,
It is necessary. Because the serum contains several molecules like proteins, cytokines, etc., that may act as variables which may complicate your end results. If you are interested, you may perform a pilot experiment with two conditions 1) Cells in 10% FBS without synchronization and 2) Serum starved cells with synchronization. You will observe a difference in the results.
Synchronization will help to get rid of the impact that the cell cycle may have on your cells in response to the treatment.
Best.
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