How to avoid central cell seeding in 6-well plates - (Apr/12/2007 )
Hi guys, sorry for the very basic question, but I can't understand how to fix this problem that seems to led to a misinterpretation of my results.
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
can't you make them suspeded or this would alter your results?
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.
I can't because when not attached they tend to clump, which is even worse.
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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.
[/quote]
This could be an idea to test. Thanks
I don't know what cell line you used, but cells might not be well resuspended before. If they can't be detached by scrapper, use TE to rinse cell twice followed by incubation for 3-5min at 37oC. Slightly flap the plate, you can see cells detached from plate. Pipetting at least 5 times to assure cells are well separated (accumulated cells tend to clump rather than individual cell).
http://www.protocol-online.org/forums/inde...showtopic=21347
hope this help.
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
How are you testing for the amount of hormone secreted?
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
How are you testing for the amount of hormone secreted?
I'm testing two different methods: RIA and chemiluminesce
Hi!
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... 