How to avoid central cell seeding in 6-well plates - (Apr/12/2007 )
This is a late answer, but maybe it'll help!
I have been told to "shake" the plate in an 8 shape.
I'll try to be clearer:
-you put your cell suspension in the well,
-pipette a little bit,
-then put the lid on the plate and
-draw an "eight" with your plate as a "pencil" and the hood as a "notebook" (
this is the clearest way I can find to tell it in english!, sorry!).Which is basically what Missele said... but in a more complicated way...
Thanks Panda for your reply. Everything clear
My problem is opposite! My cells are packed along the well wall!
You may use the above "8" shaped method.
I agree! I can confirm it works well.
Thanks to everybody
Hi eyes,
I used directly EDTA-Trypsine for two times-washing and detach the adherent cells before seeding to plates. You should check the cells during the incubation time under microscopy until the cells were well seperated individually, add the media containing serum, pippetting, seeding. This step is very important to prevent the clump of cells.
After seeding you should leave plates until cells attach to the bottle (about 15- 20 min), put in incubator.
I never shake the plate after seeding.
If you try to shake as I described, or as described panda (8 shape), you should not have your cells along the well wall, or the well is not OK.
So you should buy other plates, or maybe increase the concentration of cells in your well.
but it's true that sometime the wells are not OK.
I used directly EDTA-Trypsine for two times-washing and detach the adherent cells before seeding to plates. You should check the cells during the incubation time under microscopy until the cells were well seperated individually, add the media containing serum, pippetting, seeding. This step is very important to prevent the clump of cells.
After seeding you should leave plates until cells attach to the bottle (about 15- 20 min), put in incubator.
I never shake the plate after seeding.
I agree with you letranphan that if you triturate your cells properly then this should not be an issue. With some cell lines shaking will cause stress to the cells, which are under stress already from the trypsin and centrifugation. Single cell suspension is the order of the day!!!!!!
Hi Rhombus,
For my experience, I also never do the centrifugation of the cells after Trypsinizasion. I just add media containing surum, mix well and seed to the plates. Serum in media can inhibit the trypsin.
Good lucks
I'm working with adherent cells and I want to determine the amount of a hormone they secrete using multiwell 6.
The plates as most of them know are concave so most of the cells fall in the center. When my cells are to dense they tend to detach from the plastic altering the real results. Could somebody explain me how to avoid the "concentration" of the cells in the center of the well?
Thanks
If your plates are really concave, you have to buy other plates.
But maybe they are not really concave : when you shake the plate in a circular way, the cells tend to go to the middle. try to gently shake from right to left and then from forward to backward, or mix the cells with a pipet and don't move the plate until the cells stick to the bottom.
agree with the answer.
i think the 6-well plate is convex, not concave.
hi dude,
he post seems old , but i'll answer anyway.
I think the final solution to your problem could be the pourring of some jelly thing (like agar) down your wells to have a flat bottom.
try to find something inert where the cells will attach (not agar, because it's used to make spheroid cells that don't attach).
But to get it flat, that is pretty much the ony thing you can do.
good luck