Hello, I have non-adherent cells that I want to do mtt assay and they are suspended in media, I need a special protocol.
Hello Asma Mohammadi
Some points to remember if you are using suspension cells for MTT assay.
1. You will have to incubate cells in round bottom well-plate as it will give you a nice pellet when you centrifuge the plate. Using flat plate will result in no compact pellet and therefore there is a possibility that you might lose some cells by pipetting the medium off.
2. Centrifugation may be carried out at 1000 rpm for 5 mins in a microplate-compatible centrifuge and carefully aspirate the media.
3. Centrifugation step will be necessary to remove the treatment solution if your treatment solution is likely to interfere with the MTT color development resulting in a more purplish color. In such a case you will have to remove the test compound and rinse your cells before adding the MTT reagent.
Protocol in brief:
1. Seed cells at the required seeding density in a 96-well plate (100ul/well). Seed cells in such a way that they become at least 70-80% confluent on the day of treatment.
2. Incubate the plate at 37 degree C, 5% CO2 (unlike the adherent cells which must be incubated overnight for adherence to the substratum, suspension cells can be plated and incubated for several hours (10-12hrs.) before you start the treatment).
3. Treat the cells with the drug (at various dilutions) for the desired time points (24hrs., 48hrs., 72hrs.).
4. After the treatment period, centrifuge the cells in a microplate-compatible centrifuge and carefully aspirate the treatment solution. Centrifugation may be carried out at 1000 rpm for 5 mins.
5. (Optional step) wash the cells with PBS. Centrifuge the cells as per Step 4.
6. Add 50 µL of serum-free media and 50 µL of MTT solution into each well.
7. Incubate the plate at 37°C for 1- 4 hours.
8. After incubation add 150 µl solubilization solution to each well to dissolve formazan crystals.
9. Wrap plate in foil and shake on an orbital shaker for 15-20 minutes to fully dissolve the MTT formazan.
10. Record absorbance at 570 nm.
Best.
Hi,
did you consider to perform MTS assay? I was using it for several suspension cell lines and it worked well. Have a look on the protocols of the MTS reagent manufacturers, for example: https://www.abcam.com/products/assay-kits/mts-assay-kit-cell-proliferation-colorimetric-ab197010.html
Hi Asma Mohammadi
I would prefer to use XTT compared to MTT for non-adherent cells due to its increased sensitivity, water soluble and reading can be taken at different timepoints.
Please go through the protocol.
1. https://www.ncbi.nlm.nih.gov/books/NBK144065/
Thanks,
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