Hello Alif Rosyidah El Baroroh

When you seed your cells in a 24-well plate from a 15 mL tube, you need to resuspend or mix the cells before seeding to agitate them and generate a homogeneous solution. This way, an equal number of cells per well is ensured.

You need to first dilute the cell suspension to the desired concentration in a tube and then pipette the final volume into the wells in one step.

Cell culture media tend to foam due to the high protein content of supplemented serum. Air bubbles can hinder cell attachment during seeding and therefore create well-to-well variation of cell numbers.

So, although resuspending or mixing is necessary, you should not overdo it and avoid too much pipetting to reduce air bubble formation. Gentle pipetting also reduces shear forces and stress on your cells.

To achieve an equal cell distribution, you could use a “figure-eight” movement, or a cross-like movement of the 24-well plate. Both techniques lead to a better distribution of cells.

Hope this will help!

Good Luck.

Try to shake the plate gently for and backwards as well as to left an right in small movements. As a final step, move the plate in an "8" shape :)