How to seed the cells evenly in 6 wells plate - (Feb/04/2009 )
Thana on Feb 4 2009, 03:01 AM said:
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
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
the best way is to sip up whole media(wit unevenly distributed cells in the well) in to a tip n release them again 2 to 3 times. it wrked for me.for larger plates up n down left n rite rocking for 3 times is enuf.
-sunil kumar-
orphans on Feb 9 2009, 10:47 PM said:
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?
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?
simple pal, add the pellet to 1 ml pbs,count them in a 10 ul vol of pbs in cytometer(added equal of trypan blue), pellet them, add iml media again,add relative volume to wel.
and for info even if u know, its impossible to add xactly , its all relative n assumptive to naer tat value.
-sunil kumar-
sunil kumar on Apr 23 2010, 04:35 AM said:
orphans on Feb 9 2009, 10:47 PM said:
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?
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?
simple pal, add the pellet to 1 ml pbs,count them in a 10 ul vol of pbs in cytometer(added equal of trypan blue), pellet them, add iml media again,add relative volume to wel.
and for info even if u know, its impossible to add xactly , its all relative n assumptive to naer tat value.
The way I do it is (no need of spinning multiple times): I count the cells and do serial dilution to bring down the cell number / concentration. For example if the cell concentration is 1.5e6c/ml, then a 1:10 dilution would bring down the cell concentration to 1.5e5c/ml and so on...
S
-SciCell-
I too have been having trouble with seeding cells evenly. After reading thing and trying the tips that some of you guys have left; I am not getting beautiful even monolayers. This is what has worked for me:
In a 12 well plate; I seed my cells at the desired density then first I rock back and fourth/side to side, then do the figure eight technique (mentioned above) with the plate, and lastly let the plate sit for about 40 minutes in the hood before I proceed to move the plate into the incubator.
Thanks again and I hope this helps as well.
-Navarro-