I searched SciGine for you and found some nice methods on PBMC MTT assays. You shouldn't be removing the media. Just spec directly after adding MTT. Some people choose to lyse cells to prevent continuous metabolism of the MTT substrate as well. 

http://scigine.com/method.php?ID=55084

http://scigine.com/method.php?ID=16737

Thank you very much!! I think that it's necessary to remove the medium before tha addition of DMSO.Do you have any idea about checking PBMCs viability with trypan blue? Is the procedure the same as for adherent cells (wash cells with PBS, add trypsin and incubate for 5 min, add medium ) ?

Dear Ioanna,

I would like to present you our MTT kit that allows evaluating the viability in any cell culture format, and with any kind of cell type (adherent or suspension).

This kit comes with a very rapid and convinient protocol: very briefly, once the MTT working solution is ready, directly add it to suspension cells incubate at 37°C and at the end of incubation add the solubilization solution.

once the converted dye is completely dissolved measure the absorbance at 570nm (you can find more details on the protocol in the following link).

This is thus a real alternative that you could try. Please do not hesitate to contact me via ResearchGate or directly at : [email protected] if you need more information.

best regards,

Cedric

http://www.ozbiosciences.com/assay-kits/51-mtt-cell-proliferation-assay-kit.html

Hello Ioanna Elef, In this moment, I´m trying to perform the same assay to test PBMCs with some essential oils. I wonder if your cells support during this time of incubation or die before adding the MTT.

Thank you very much and thank you for your attention, collaboration and answer.

Elizabeth

Hi Eli Quintero,

I have done many times the MTT assay and all the times, cells were alive. I always used to check the cells under the microscope before the addition of MTT and four hours after the addition of MTT I checked the crystals under the microscope! But in order to be sure I counted the cells with Trypan blue too, just to confirm my results. Do you have any problem with cell viability ? Do you culture the cells for a long time after the isolation ? 

u can just spin the cells down in plate centrifuge, before removing the media and then proceed with MTT.

Live-Dead analysis of Cells can be done by using Fluroscein Di Acetate and Propidium Iodide very easily

Dear Ioanna,

an article was just published using our MTT kit for quantifying proliferation in Jurkat T cells.

maybe you could refer to it for more information, please see the link below

best,

Cedric

http://online.liebertpub.com/doi/abs/10.1089/nat.2016.0624

Can we culture the PBMC cells for a long time without any extra additions？ I used to think that PBMC must be used after the isolation ?  If not, the cells will be died.

Article Optimization of Human Peripheral Blood Mononuclear Cells (PB...

Can you send me the reference that you are using in this test.

Thank you.

Can anyone please tell me approximate absorbance value of PBMCs at 570 nm? I know it may vary, but I just need an approximate value.

Aditi Mahajan approximately 0.185.

Thank u @ Md. Mahmudul Hasan will definitely look into it

Hello Loanna.

How are you?

Did you find or formulate any protocol?

I'm also interested!

Hi Caique Figueiredo the experimental procedure I apply is the following: after isolation and count of PBMCs, I seed 100μl of them (100000 cells/well) and 100μl of the tested extract->incubation 24hours. After this, I add 20μl MTT-> incubation 4hours. I always check for the presence of formazan crystals under microscope. Then, I centrifuge the cells, remove the media carefully and add DMSO. Good luck!!!

Thank you Ioanna Eleftheriadou !

Toxicity measurements can be taken even by not removing the cells and medium before adding DMSO.Many workers do that.If medium is to be removed then the microplate should be centrifuged first to prevent the loss of cells

Thank u mam @ Asha khanna

How do one add DMSO without removing MTT solution?

Ioanna Eleftheriadou Actually the cells and test compound should not be added together. After isolation, the cells require some time for attachment. Moreover, the cells will be stressed when you are adding test compounds soon after isolation. Usually, we are allowing cells to attach for 24h and later challenge with a test compound for a required time period. Afterwards remove media and rinse cells with PBS and incubate with MTT solution for atleast 4h. Remove MTT after 4h and dissolve crystals with DMSO. Read OD at 570. This is the standard protocol as per ISO 10993-5, Annexure

hai Jolly, try MTS reagent. you don’t have add DMSO to measure the cell survival. The byproduct is water soluble and you can take the reading directly after 4 hr incubation.

You might want to use PrestoBlue its quicker and less cytotoxic to the cells.

Ioanna Eleftheriadou If you centrifuge the plate after the 4h incubation and remove only the supernatant by pulling with the pipette close to the well wall, the cell removal is minimal. Also, we use 500000 of PBMCs per well. We consider the absorbance with 100000 cells too low. The value you quoted (0.185) is too low considering the detection limit of MTT. With this absorbance, the compound may appear more cytotoxic than it really is. The ideal is values closer to 1. With 500000 cells per well, it is ~0.800 at 540nm.

Thank you sooo much for your input