How to seed the cells evenly in 6 wells plate - (Feb/04/2009 )

Hello,

I need to seed my cells (4.5x106 cells/well) in 6 wells plate and let it double over night to reach the concentration of 9x106 cells/well.

However, when cells have doubled overnight, they don't attached to the wells but floating around the wells which make the concentration wrong.

The floating ones are not dead at all but not attacthed.

Does anyone know any techniques or how to make them attached nicely after the doubling??

Any suggestions would be appreciated,

Thanks

if attachment is a problem, coat plates with poly lysine or something similar. Are the wells confluent the next day?

Here are some very good discussions on this topic:

How to avoid central cell seeding in 6-well plates
How to seed cells evenly in small well plates

what cells are they? I had some issues with HT29 cells that they didn't attach as fast as HeLa cells, but if I left them for 1-2 days they spread all over the flask or 6-well plates.

It depends on the cell type but I suspect your cells might be dead. how do you say they are not dead? did you try checking them for Trypan Blue uptake?

do you know what trypan blue uptake is? if not I will explain to you in the next post.

hi!

Have this research project on cell culture and am having problems on how to seed 3, 000 cells per well in a 96-well plate and make sure that its 3,000 cells adhering to the well. Any suggestions?

hi!

Have this research project on cell culture and am having problems on how to seed 3, 000 cells per well in a 96-well plate and make sure that its 3,000 cells adhering to the well....Any suggestions?

1) when adding cell suspension, tilt the plate at 45 degree and add 2ml of cells to the lower side of the wells, then place the plate level without any other movement. Usually this will be enough to seed the cell evenly.

2) if not, swirl the plate clockwise and anticlockwise 3 times each, back and forth, right and left 3 times, return to incubator, after 5 min, check the plate, if not evenly, repeat that again.

Thank you so much for the links, I've tried with all the suggestions and the cells attatch nicely now

There were so many good advices and techniques on those links, Thanks a million bioforum and all replies

I really appreciate your helps

Thana, which techniques did you end up using? I seem to be having the same problem and it is throwing off my numbers.

you seeded too many cells in one well.