Hello Shadi Hambo

You may incorporate the following in your protocol, besides controlled humidity (protection of wells drying out), the non-disturbance of the plate/cell layer, and pipetting.

1. Pre-incubate the plate with media for a short period before adding cells as it can help stabilize the temperature and gas equilibrium, potentially encouraging more even cell distribution upon seeding.

2. Regularly check and adjust the incubator shelves to ensure they are perfectly level, preventing cells from drifting to one side.

3. When adding the cell suspension, distribute it slowly and across multiple areas of the well instead of focusing on a single spot. Using a criss-cross pattern can help.

4. Minimize vigorous shaking and ensure slow, even movements of the culture plate before and during the initial cell attachment phase.

5. You may use a uniform cell seeder, if you have one. It is a device designed to achieve a consistent and even distribution of cells across a culture surface during cell seeding. It works by confining a cell suspension within a specific height above the culture surface, preventing uneven distribution caused by fluid flow or other factors.

Hope these suggestions help!

Best,

Dear Malcolm Nobre,

Thank you very much for the suggestions and tips.

I will take them into consideration for the upcoming experiment.

Best,

Did you notice the uneven distribution before infecting cells?

Hi Shadi

Maybe you can leave the outer wells and fill them with PBS? It once worked for me.

In my experience, I encountered this issue when attempting to evenly distribute the cells by shaking the plate, which often caused them to aggregate in the center.

What worked for me was applying minimal movement—just one or two gentle motions side-to-side and up-down—sufficient to spread the cells without overmixing. After that, I allowed the plate to rest on a flat surface, either on the lab bench or directly in the incubator (not necessarily inside a biosafety cabinet). This approach consistently gave me even distribution.

Hope this helps!

I agree with Narendra Kumar ; prepare a solution of medium with the right concentration of cell; for P96 +/_40 000cells/200ul) and dispense (200ul/wells in the P96 wells with a multi channel pipette; don't shake it/ put in the CO2 incubator right away

for P6 once you have placed the plate on the incubator shelf you may shake them twice slowly doing a "cross" NOT a circle