Hi, all.

MTT or CCK8 experiments can be performed directly in 96-well plates.

After culturing cells in 6-well plates, trypsin digestion and then transfer to 96-well plates for MTT experiments is theoretically possible, but both trypsin and MTT damage cell viability. You can let the cells recover for 2-6 hours after digestion, and then add MTT after the cells adhere to the wall.

You can refer to the following MTT method experimental steps:

1. Preparation of MTT working solution

Dissolve MTT in PBS to obtain 5 mg/mL MTT.

2. Cell proliferation detection (96-well plate)

2.1 Inoculation of cells: Prepared single cells are used to prepare suspensions with culture medium containing 10% FBS, inoculated in 96-well plates, 1000-10000 cells per well, and the volume of each well is 100 μL.

2.2 Cultivate cells: 37℃. 5% CO2, incubate for 24-72 h.

2.3 Add 10 μL MTT to each well, incubate for 4 h, discard the supernatant, and the suspended cells need to be centrifuged first.

2.4 Add 100 μL DMSO and shake for 10 minutes to completely dissolve the crystals

2.5 Use an absorbance plate reader to monitor the increase in absorbance at OD=562nm.

The above is organized by the MCE technical team and I hope it will be helpful to you

The answer to this question comes from MedChemExpress (MCE) Technical Support.

[email protected]

Hello Lucian Miles

Why do you need to use a 6-well plate?

If you are planning to test the effects of different drug concentrations on OSCC cells using the MTT assay, you may grow the cells in T-25 cm^2 / T-75 cm^2 flasks and then trypsinize the cells on 80% confluency, and seed them in 96-well plate as the per required density per well such that they become 80% confluent in 24 hours. After 24 hours when the cells have attached to the substratum, you may treat them with different drug concentrations for different time points namely, 24, 48 and 72 hours. After each time point, you may carry out the MTT assay as you have mentioned in your protocol.

I have made a mention of this because T flasks are much more economical and easy to handle than 6-well plates. They are designed for large-scale cell culture due to their higher surface area and larger volume capacity. For planning your experiment, you will require lot many cells and so culturing them in flasks (T25cm^2/T75cm^2) is a much better idea than a 6-well plate.

Best,

It is technically possible to culture OSCC cells in 6-well plates, then trypsinize and transfer them to 96-well plates for MTT assay, it is not recommended due to variability and poor reproducibility. The MTT assay is sensitive to initial seeding density, cell cycle state, and attachment, and transferring cells introduces inconsistencies in cell number, stress from trypsinization, and delayed reattachment, which can significantly affect metabolic readout.Instead, for accurate and reproducible results, you should directly seed OSCC cells into 96-well plates, allow them to attach and reach exponential phase, and then apply drug treatments. This ensures uniform growth and better interpretation of dose-dependent effects.

I have never done such an experiment before, but it may succeed or fail depending on the safety of the cells and the cleanliness of the work so that no contamination occurs.

Yes, cells cultured in 6-well plates can be successfully trypsinized and transferred to 96-well plates with proper technique to ensure cell viability and uniform distribution. The process involves first aspirating the medium from the 6-well plate and gently rinsing the cells with PBS to remove residual serum that could inhibit trypsin activity. Then, an appropriate volume of trypsin-EDTA (typically 0.5–1 mL per well) is added to detach the cells during a 2–5 minute incubation at 37°C. Once cells are detached, the trypsin is neutralized by adding complete medium containing serum, and the cell suspension is pipetted gently to achieve a single-cell population. It is crucial to count the cells using a hemocytometer or automated counter and dilute the suspension to the optimal density for the 96-well plate, which generally ranges between 5,000–50,000 cells per well depending on the cell type and experimental requirements. The cell suspension is then carefully transferred into the 96-well plate, typically using 100–200 µL per well, and the plate is gently swirled to ensure even distribution. For adherent cells, a 24-hour incubation period is recommended to allow proper attachment before any treatments or assays, while suspension cells can be used immediately after seeding. To maintain consistency, edge wells prone to evaporation should be filled with PBS or left unused, and all steps should be performed under sterile conditions in a laminar flow hood. This method may not be suitable for cells highly sensitive to trypsin, in which case alternative detachment methods like scraping should be considered, or for experiments requiring extremely precise cell numbers, where direct seeding or cell sorting might be preferable. For optimal results, cell counting and seeding density should always be optimized for the specific cell line and experimental conditions. Let me know if you require further assistance with protocol refinements or troubleshooting.