Hello Mr. Sayed, Trypsinization and subsequent transfer may lead to uneven seeding, cell loss, or stress, which can potentially affect assay results. Furthermore, trypsinization and handling may compromise cell viability, thereby interfering with the accuracy of the MTT assay. In my opinion, culturing and treating cells directly in 96-well plates is the preferred approach to reduce variability and align with standard assay protocols.

However, if resource limitations necessitate an alternative approach, certain optimizations can help. Ensure uniform trypsinization and use a cell counter to normalize cell density before seeding into 96-well plates. Minimize cell stress by working quickly and employing gentle pipetting techniques. Validate the method by comparing results with the standard protocol, assessing reproducibility, and confirming cell viability through alternative assays. These measures will help mitigate variability and ensure reliable MTT assay results.

I agree with the comment of R Habib, that tryppsinisation and subsequent transfer will lead to uneven cell density, you have probably interest to grow and treat the cells in the same plates. If you use a spectrophotometer, It will be perhaps interesting to use either 12 or 24 wells plates. It is easier to monitor cell density and medium evaporation in such plates to avoid the great variability observed in 96 wells plates.

I usually do my MTT assays in 96 well plates and test drugs too. I suggest you to be carefully in the number of cells you seed before drug treatment but I dont believe that trypsinitazation is the best option . It would affect your cells viability

Reyna Marie Sanchez

thank you all for the helpful responses! I do have some other questions

I am working with oral cancer cells, which adhere to the container surface. Lets say I am keeping them in a 6 well plate and adding drugs to the same plate. Could I keep them suspended in the 6 well plate by occasionally (gently) pipetting them in the medium, so that they do not adhere? Then after the incubation time points (24, 48, 72 hr) I can transfer them (cells + drug) to a 96 well plate just for the mtt assay?

Hi Omama,

It should be noted that the cells respond differently in 3D (suspended) and 2D (adhered) state. Pipetting may not be a good method to carry the experiment thinking the cells are uniform and healthy, they can undergo stress at anytime due to agitation. So, generally this is not recommended. Most of the time the drug is tested when the cells are adhered, in case of adherent cells as I could get from the steps you intend to perform. You should allow the cells to adhere before treatment and mention that in your protocol. Post incubation, transfer them to 96 well plates, I would suggest doing intra and inter well plate variation check by withdrawing equal quantity form each well and analysing the absorbance intensity from the MTT assay for any variation. Say, you can distribute uniform quantity of one well of 6 well plate into 6 wells of 96 well plate, take readings and repeat the experiment at least thrice.

Hi Omama,

The MTT or CCK8 assay can be directly performed in a 96-well plate. Alternatively, you could also culture cells in a 6-well plate, followed by trypsin digestion and transfer to a 96-well plate for the MTT assay. This is theoretically feasible. However, both trypsin digestion and MTT assay can affect cell viability. Therefore, after trypsinization, it is recommended to let the cells recover for 2-6 hours until they fully adhere, before adding the MTT reagent.

Here is a reference MTT assay protocol for your consideration:

1. Preparation of MTT working solution

Dissolve MTT powder in PBS to prepare a 5 mg/mL MTT solution.

2. Cell proliferation assay (96-well plate)

2.1 Cell seeding: Prepare a single-cell suspension in culture medium containing 10% FBS, and seed cells into a 96-well plate at a density of 1,000 to 10,000 cells per well, with a total volume of 100 μL per well.

2.2 Cell culture: Incubate at 37°C, 5% CO₂ for 24 to 72 hours.

2.3 Adding MTT: Add 10 μL of MTT solution to each well, incubate for 4 hours, then remove the supernatant. For suspension cells, centrifuge first before removing the supernatant.

2.4 Dissolving formazan crystals: Add 100 μL DMSO to each well, and shake for 10 minutes to fully dissolve the crystals.

2.5 Measuring absorbance: Measure the absorbance at 562 nm using a microplate reader to assess cell proliferation.

This protocol is provided by the MCE Technical Team Binbin Lee — hope you find it helpful!

Thanks for Dr. Miles, our technical support team also provide specific product Protocol and User Guide in website, here's the link: https://www.medchemexpress.com/MTT.html