How to seed adherent cells evenly in 150mm dishes? - (Jul/23/2007 )
As the title, I've never successfully done that.
Any trick? buddy
Thanks!!
1) make sure the incubator is placed level.
2) Put enough medium to the dish.
3) avoid any bubbles.
4) mix cell and medium well
One more important thing:
After adding the cell suspension in the dish, _do_not_swirl_it_! The more you try to homogenize it, the more they accumulate in the middle of your dish. So just let them lie flat in the sterile hood for 20 mins (go have a coffee). This is enough time for the cells to attach a bit. Then transfer them into the incubator. Could U please let me know how it worked for you? It worked very nicely for me in 60 mm dishes, you could clearly see the difference!
yes, never swirl it. if you want to distribute it evenly but not swirling it... you can try this: after adding the cell suspension, homogenised in horizontal direction -- left and right direction.
Hm. I'd say don't even do that. But maybe it is already the field of religion, and not science.
It would be interesting to see if you tried it with both (doing nothing and horizontal mixing), and then look at the difference. you could try moving it left -right and forward and backward. It may help.
after placing the culture dish in the incubator, take care while closing the door, as this may create ripples in the dish and make the cells to move.
We routinely use 60mm petri dishes for clonogenic survival assays. The trick we use to distribute the cells evenly on the plates is to gently shake the cloning box left to right and front to back before placing it in the incubator. Avoid swirling the box as this will end up with clumping in the centre of the plates.
Thank you so much!
I tried horizontal mixing(left, right, forward and backward), then the cells are evenly distributed!
Maybe the trick is: NO SWIRL, MIX HORIZONTAL(preferably in 2 or more perpendicular directions).
Genehunter's advices are very precise and comprehensive.
Scolix's carefulness is surprising to me.
After adding the cell suspension in the dish, _do_not_swirl_it_! The more you try to homogenize it, the more they accumulate in the middle of your dish. So just let them lie flat in the sterile hood for 20 mins (go have a coffee). This is enough time for the cells to attach a bit. Then transfer them into the incubator. Could U please let me know how it worked for you? It worked very nicely for me in 60 mm dishes, you could clearly see the difference!
I tried your method. It doesn't work for me in 150mm plates.
I don't know why. But still thanks!
I found the easiest way to do this is to add suspended cells to 30-35 ml medium in a 50 ml centrifuge tube, mix well, pour it into the plate and place the plate in a LEVELED incubator. It has been working very well for me.