6 replies to this topic

[Ana G]

Hello everyone!

I'm planning on using the MTT assay to evaluate the viability of my cells (THP-1 and PBMC) after treatment with some compounds. I've never performed this assay before so I collected some protocols but I'm still very confuse, so I would appreciate if someone could answer me a few questions:

1- My cells are in suspension. I don't know if I should treat them like that or if I should try to attach them by making a previous incubation. The problem is I don't know if they attach at all or if their behavior changes if they become adherent. On the other hand if I use them in suspension how am I suppose to remove the media before I add the MTT solution? Some refer a centrifugation step but I don't know how this can be done in a 96 well plate!

2- Some refer removing the media and wash after treatment with the compound and with the MTT solution while others don't. Are these steps necessary? (again the centrifugation problem!!!!!)

3- To dissolve the crystals, some use DMSO, others isopropyl alcohol/HCl, and others SDS/HCl. I don't know which solvent to choose.

(If an answer come from someone who works with my cell type please refer it)

Thanks very much in advance.

[zienpiggie]

1. If they don't attach to the plate, then you can add MTT reagent in a more concentrated form to the media to make the final concentration in the wells itself. If you need to remove your treatment you may need the centrifugation step. It likely refers to plate centrifuge which is like a regular centrifuge but for plates.

2. It depends if your test compound interferes with MTT assay. If your treatment has strong antioxidant properties, it will interfere with MTT assay when treatment is mixed directly with the MTT reagent since the assay is based on the redox activity of mitochondrial succinate dehydrogenase.  You may thus need to remove your treatment prior to MTT addition.

3. SDS/HCl works fine.

[Ana G]

zienpiggie, on 09 December 2011 - 12:21 PM, said:

1. If they don't attach to the plate, then you can add MTT reagent in a more concentrated form to the media to make the final concentration in the wells itself. If you need to remove your treatment you may need the centrifugation step. It likely refers to plate centrifuge which is like a regular centrifuge but for plates.

2. It depends if your test compound interferes with MTT assay. If your treatment has strong antioxidant properties, it will interfere with MTT assay when treatment is mixed directly with the MTT reagent since the assay is based on the redox activity of mitochondrial succinate dehydrogenase.  You may thus need to remove your treatment prior to MTT addition.

3. SDS/HCl works fine.

zienpiggie,

Thank you for your answer. Indee my treatment has antioxidant activity, so I guess I will have a problem here. I don't have those centrifiges for 96 well plates. I didn't get if it is necessary to remove the MTT solution before adding the mixture to dissolve the crystals. Can you clarify this doubt?
Thanks again!

[zienpiggie]

Hi Ana G,

Once you added your MTT solution to allow the crystal formation, you do not need to remove the MTT solution. Just add SDS/HCl straight to the wells. What you may need to remove is your treatment solution prior to addition of MTT solution, since if they are combined together, they will interfere with the MTT color development resulting in a more purplish color. You can however test if your compound will interfere with MTT assay by mixing your test compound + MTT solution in the absence of cells. If they still turn purple as opposed to control, then you have interference. If this is the case, and there is no way that you can remove the test compound and rinse your cells before adding MTT, then may be looking into other viability assay/proliferation assay is a good idea.

[ReemooN]

hey,

I would like to know after getting the absorbance how the calculation will be ??
and what should the positive control be ?

thanx

[fraffly]

If you culture the cells in suspension, keep it that way for the assay. Centrifugation doesn't bother these cells much.
For suspension cells, I'd incubate in round bottom well-plates, then you'll get a nice pellet when you centrifuge them. In flat plates no compact pellet can be achieved and you might loose some cells by pipetting the medium off.

- centrifuge (for PBMCs 300g/5min works well)
- remove medium
(opt. make a washing step)
- add MTT solution, resuspend cells, incubate
- dissolve crystals, I've used DMSO, it works fine. If you're in doubt, just test the different solutions.

[Jen55❤]

I also have a question about which plate type to use. I am doing a cytotoxicity assay on suspension cells. I will be putting the plate in a plate reader, I won't need to centrifuge them. So, if I don't need to spin the plate, does it matter if I use flat bottom or round bottom plates? I can also set the program to shake so the cells don't settle.