How to seed cells evenly in small well plates - (Oct/20/2006 )
hi all,
I am a new one with cell culture work. I meet one problem when seeding my cells on 24well plate. Most of the cells focus on the center lead to overgrowth while there are very litle cells on the surounding area. I meet this problem many time, and I has try to touch the edge of wells when pipeting or seed directly at the center(dont touch the edge of well), tap the plate after seeding or immediately put in incubator, pipeting gently or strongly...But not any difference. I dont know the reason of this problem and how to deal with it.
Please suggest me some idea to deal with this problem.
Thanks .
What I do is that I shake it gently from left to right, and from far to close (horizontally), and cells are well dispersed (don't make circles when agitating)
But sometime the wells are not flat, and you can't do anything.
I am a new one with cell culture work. I meet one problem when seeding my cells on 24well plate. Most of the cells focus on the center lead to overgrowth while there are very litle cells on the surounding area. I meet this problem many time, and I has try to touch the edge of wells when pipeting or seed directly at the center(dont touch the edge of well), tap the plate after seeding or immediately put in incubator, pipeting gently or strongly...But not any difference. I dont know the reason of this problem and how to deal with it.
Please suggest me some idea to deal with this problem.
Thanks .
in addition to the other advices:
reduce the number of cells to seed; pipet them dropwise at various sites; maybe lack of proper adhesion sites, so cell-cell adhesion is perfered; try different coats (collagen, polylysine, fibronectin, laminin, vitronectin, matrigel)
we tilt the plate nearly 90 degrees and making sure the media isnt flowing out of the chambers ( they usually dont) for 10 times both sides (left to right and forward to backwards). This helps a lot.
i find if you let the cells attach in the hood for a short while before moving to incubator its better . . .. because if you have to walk to the incubator, you create a swirling motion in the wells as they are round, which draws the cells into the centre . . . .i leave mine sit in the hood for about 10 min after seeding and it has helped a lot. you should try and see if it helps
yes, I confirm avalon - when they build their filopodia cells should not be disturbed, which means directly after seeding; 10 min may be prolonged to 30 min; it depends on your type of cells
I have had a similar problem also and I found that increasing the media volume per well can help, not quite sure why, I was told something about surface tension.
Also agree with the others, that leaving for 10 or so minutes to attach before moving to the incubator can help.
Alternatively, you can sit your plate on a plate rocking platform at a very slow setting for a little while.
Thanks for all your susggestions. I has tried to shake the plate after seeding, pipetting droplet at different position, reduce the number of cells. But, no any difference I will try the other idea.. I hope one idea here will help me.
agree with lauralee
adding more medium always works for me
of course you should shake the plate before putting into the incubator
what bother me is that the inner of our incubator isn't even, i mean the board in the incubator( i don't its name,sorry, hope you could understand) , one side of the flask always have more cells and the other have little , and i can do nothing.
anyone can help me?
thanks in advance
I've had a incubator shelf that was like this.. shove a piece of plastic or metal under the rack on the lower side until it's level.
As for even plating... especially with small wells, just walking the plate over to the incubator can make the media swirl and push cells out the the edges. I always put my plates on the rack in the incubator and then give them a few good shakes back and forth to re-distribute cells evenly.