12 replies to this topic

[distazio]

: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

[pcrman]

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.

[distazio]

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:

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.

Attached thumbnail(s)

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[distazio]

more similar at this another image that i upload

distazio, on May 15 2009, 10:44 AM, said:

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:

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.

Attached thumbnail(s)

• 

[distazio]

i have taken an image after trypsin of my ES clone...what happened?? help me please ..is it contamination or EB after tripsin treatment???

Attached File(s)

•  HEK_B5023.tiff (577.25K)
  Number of downloads: 62
•  HEK_B5024.tiff (577.53K)
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•  HEK_B5025.tiff (577.28K)
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[Sandy_8]

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?

[distazio]

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??

[miRNA man]

Puro selection is usually very fast, much faster than G418. Why do you need 20-30 days?

[distazio]

shure. i don't know why but the clones grew slowly the first 10 days.

but usually i select with puro in 10 days.

[Bill Nye]

Sandy_8, on May 18 2009, 11:47 AM, said:

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?

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:

shure. i don't know why but the clones grew slowly the first 10 days.

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.

[enock02]

hi guys, do the big colonies are a monolayer of cells or they like the cancer cell, usually form focus?

[Radish]

enock02, on 03 October 2010 - 12:40 AM, said:

hi guys, do the big colonies are a monolayer of cells or they like the cancer cell, usually form focus?

Hi bioforumers
I don't know which big colonies you are referring to, but, the "translucent" ones don't seem to be birefringent so they are probably a monolayer, the brown ones are definitely a 3 layer structure.

What do you mean by focus?

[stemcellconclave]

 distazio, on 18 May 2009 - 03:15 PM, said:

shure. i don't know why but the clones grew slowly the first 10 days.

but usually i select with puro in 10 days.

But the embryonic stem cell technique is save or not. I have heard something different about this technique.

Stem Cell Conclave Event & Exhibition 2011