For my research project, I often have to seed cells into cell culture plates for cytotoxicity assays like SRB and MTT. I have noticed that whenever I seed cells into the wells of a 96-well treated flat-bottomed cell culture plate the cells accumulate in the corners. Is there any way I can evenly spread the cells across the bottom of the well?
(I am concerned because I think I would get more accurate absorbance values if the cells were spread evenly across the wells)
Full disclosure; I am seeding NTERA-2 cells, I used a Nichiryo 20 µL - 200 µL micropipette to seed. The photo has about 10,000 cells per well. The photo was taken about an hour after seeding which is why the cells are still spherical. (Sorry about the photo's poor quality but it is good enough to see that cells have accumulated on one side).
Hello Navodya Silva
To achieve an even distribution of cells in the well for a 24-well plate, I make use of figure eight-like movement, while one may also use a cross-like movement of the plate. Both these techniques lead to a better distribution of cells in the well.
However, for wells of smaller diameter such as the wells of a 96-well plate, the movement of the liquid is less, leading to an uneven distribution of cells. An effective way to circumvent this effect during cell seeding would be to first dilute the cell suspension to the desired concentration in a tube and then pipette the final volume into the well. Do not add the cell suspension to the well already containing the media for dilution.
Moreover, do not shake the plate after seeding the cells as this shaking movement may move the cells to the edge of the well. A little vibration while walking with the plate from the laminar hood to the CO2 incubator may also cause uneven distribution of cells in the well.
So, I suggest after having diluted the cell suspension to the concentration you desire, and after having pipetted the final volume into the well, you may use a tray to transfer the 96-well plate form the biosafety cabinet to the CO2 incubator.
Best.
Pipetting the cells in the middle of the well will push them to the edges due to the pressure exerted on the center of the well, you can try and pipette around the edges of the well by tilting the pipette and moving in a slow circular motion to cover the entire circumference of the circle.
I also used the same technique that Malcolm recommended, worked OK for me.
I agree with Malcolm's answer, do not make shaking movements to the 96 well plate after seeding and you may try to drop the cell suspensions that have already diluted into the desired concentration right in the middle of the well plate, drop them gently.
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