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help with cell seeding - (Aug/23/2011 )

ive been having issues with seeding HeLa cells in 24well and 6well plates.

For example:
- i use trypsin/versene to trypsinize my cells
- take 100ul of the cells and dilute it 1:10
- count my cells with hemacytometer
- then i calculate the cells required for seeding and i always make sure to add a little bit more to make in case of pipetting error/cell death (our lab doesnt use trypan blue)

then i seed my cells at 50% confluency then the next day my cells are not even 90-100% confluent
they are are pretty much 20-50% confluent; i dont know wats going on. wat am i doing wrong????

i tried mixing the cells before i seed
i tried using less trypsin
but they havent worked

the only way my seeding works is to supersaturate the wells with cells and aspirate cells that havent adhered :(


Do you leave them a full 24 hours or is it shorter?

Cells, like any organism have what is known as a lag phase where they take a while to start replicating in log phase again after seeding, this is usually due to low density and the time taken to adapt after seeding (i.e. re-attach etc.) You could try seeding at a higher density if you need 90-100% confluent.

It could also be that you have had your cells up for quite some time and they are now not behaving as they normally would. Ideally you would get fresh cells up about every 10 passages.

It may also be that you are using the haemocytometer incorrectly or underestimating the number of cells need to give 50% confluent.


Agree with bob1. Cells do need some time to "pick up" the momentum to grow.
I used to grow some cell lines where after the cells attached, they don't really proliferate straight away. They would take another day to double. So if I need certain number of cells the next day, I would seed exactly the quantity I need.

Maybe you can do a simple proliferation assay. Then you'll have a good idea of how long for your cells to attach and doubled.
Also, you'll know how long for your cells to get 90% confluency if you seed certain cell number today.