Hi, I recently did an MTT assay using NIH 3T3 fibroblasts. I seeded 10,000 cells/well, let them grow for 24 hours, and added my drug/chemical after 24 hours. However, I think the cell count was too high for fibroblasts since the cells were pretty confluent on Day 3 and started detaching from the periphery. I would recommend using either 5000 or 2500 cells/well to see which works better.

Dear Noor,

Make a serial dilution of different cell concentrations ex: 5000, 10000, 15000...

Take the optical density of them and plot your data, then decide the one that fall in the exponential range of the chart.

Dear Noor,

Make a serial dilution of different cell concentrations ex: 5000, 10000, 15000...

Take the optical density of them and plot your data, then decide the one that fall in the exponential range of the chart.

Mohamed Khashan thank you for your kind advice. Since I am new to cell culturing so should i measure the cell density immediately after the dilutions or after incubation for 24 hrs?

How can I determine the exponential range? Can you suggest any article related to this?

Thanks again

Pls refer to the following link it has a detailed explanation:

https://www.mdpi.com/1422-0067/22/23/12827

Mohamed Khashan thank you!

Dear Noor

I performed MTT experiments with hydrogels with 3T3 fibroblasts, I normally seed 8000 cells/ well in 96 well plates.

Yousof Farrag did you use the extract of hydrogels or seeded cells directly on the hydrogels?

I tried different ways of contact between the cells and the hydrogels. In general I preferred the direct contact as more realistic approach for my application.

I tried seeding the cells and the adding the hydrogels, seeding the cells on the hydrogels, mixing the hydrogels with the culture medium (injectable hydrogels), as well as homogenizing.

You should choose depending on the application, the cells, the nature of your hydrogel.

You should be aware of the amount of the hydrogel you add not to alter too much the nutrients concentration of the culture medium.

Yousof Farrag thank you for your help!