Central cell seeding - (Aug/30/2009 )

Hi,

I kept on having the problem of central cell seeding on two cancer cell lines when seeding the cells in 24 well plates. Before, seeding the cells, I tried to dislodge the cell pellet by flipping the falcon and pipette up and down several times. The cells were separated into individual cells when I did the cell counting by a haematocytometer. After seeding the cells, I tapped the plates gently/rocked the plate back and forth to make sure the cells were evenly distributed and waited for 5-10 mins to let the cells settle down. I observed the cells under a microscope and found most of the cells were distributed in the centre of the wells. Then, I repeated to do the tapping/rocking the plate again and put the plate aside and waited for 5-10 mins again. The same thing happened again!!! (P.S. my 24 well plates are flat-bottomed)

Are there some cell types having the tendency to move towards each other? Because this really affects my transfection experiment, what should I do to solve this problem?? Has anyone encountered this prolem???

Thanks!!

Samantha

The manner in which you rock the plates/slides can have an effect.
If you use a circular motion then you will concentrate cells in the center of the plate as preference to a forwards backwards motion.

I don't know if that might be your problem.

I'm not really sure if its to do with the cell characteristics. With peripheral blood lymphocytes U had the same thing. This didn't matter because they are suspension cells.

I think its to do with the manner of shaking, the wells are round so even if you use a linear movement, fluid will still "slosh" around the well. Maybe light pipetting will help for mixing instead of tilting?

I would be very interested to see if there is a reason, I didn't really think to much of it.

Unfortunately there isn't much you can do about the higher density in the middle. It is purely down to size of the well creating a bigger vortex which causes the cells to aggregate in the middle of the well. Could you try doing your transfections in 6 well plates, or with lower cell numbers to help minimize this effect?

We have found that adding a bit more media can sometimes help.

That, and taking the plates out of the hood soon after aliquoting in the cell/media mix- the base of the hoods are not level, and the vibrations can also affect where your cells settle. This was much more noticable in our old hood (we had to take the plates out as we made them, 1 by 1, which is a pain in the bum when you are setting up 60 odd!!), but not really a problem in our new hood.