13 replies to this topic

[Thana]

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

[scolix]

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

[bioforum]

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

[Curtis]

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.

Edited by Curtis, 04 February 2009 - 06:50 AM.

[orphans]

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?

[orphans]

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?  

[pcrman]

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.

[Thana]

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

[Eagles09]

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

[frankfan]

you seeded too many cells in one well.

[sunil kumar]

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

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.

Edited by sunil kumar, 23 April 2010 - 03:41 AM.

[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?  

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.

[SciCell]

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?  

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

[ACN]

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.