Embryonic bodies? - (May/14/2009 )
:blink:hallo
i need an help.
i'm working with mice ES cells. i have electroporeted the cells with a costruct that carring puromycin resistence.
after several days ( 20-30 days) of puromycin selection, i have seen strange clones in the dish. 
the clones were very big and difficult to disperse. Very strange to me. I was wondering whether it is a EB. is it possible even if i'm working with LIF in the medium??
thank's
EB is usually formed in suspension culture and is round shaped cell sphere. I don't think the big clones are EB. Have you tried mechanic methods of dispersing the clones? upload an image if you can.
the clones are in adhesion and is very difficult to disperse the clones with tripsyn.
the image is very similar to this one that i have fund in the web.
pcrman on May 15 2009, 05:21 AM said:
more similar at this another image that i upload
distazio on May 15 2009, 10:44 AM said:
the image is very similar to this one that i have fund in the web.
pcrman on May 15 2009, 05:21 AM said:
i have taken an image after trypsin of my ES clone...what happened?? help me please ..is it contamination or EB after tripsin treatment???
Simply...They're overgrown....and they became like embryoid bodies....
you must split them before they reach this size...
Have you splitted them during the selection?
ahiahiaiii no i haven't split them!! and indeed the selection has been very long..(20 days)
ok and now?? can i work with this cells??
Puro selection is usually very fast, much faster than G418. Why do you need 20-30 days?
shure. i don't know why but the clones grew slowly the first 10 days.
but usually i select with puro in 10 days.
Sandy_8 on May 18 2009, 11:47 AM said:
you must split them before they reach this size...
Have you splitted them during the selection?
Those are not EBs. Sandy's right, your cells are simply over confluent. I think you can revive them, trypsinize and reseed... they should be able to recover.
distazio on May 18 2009, 07:15 PM said:
but usually i select with puro in 10 days.
If your initial seeding density is low, the cells will take longer to grow. If they are adherent cells, they need to first adhere to the growth surface before they begin to grow... so that may explain the extended lag phase that you experienced. Mycoplasma may also reduce growth rate.