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Help! ES Lenti infection efficiency=nightmare


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8 replies to this topic

#1 Radish

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Posted 25 January 2011 - 10:54 AM

Hello bioforum!

Please help me!
I am infecting ES cells with Lentis carrying shRNAs but I haven't been able to get more than 40% of the cells infected.
I concentrate the virus by ultra centrifuging them (with PEG it the efficiency was down to 20%). I do the infection with the cells in suspension and it still sucks.
If you guys have any thoughts on how to improve this please share them with me. I am totally stuck!

#2 Clare

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Posted 26 January 2011 - 03:23 AM

Hi,

There could be many things that are going wrong !

Are you adding polybrene?

Have you tried transducing another cell type with the same virus? Is the efficiency bad too?

Clare

Hello bioforum!

Please help me!
I am infecting ES cells with Lentis carrying shRNAs but I haven't been able to get more than 40% of the cells infected.
I concentrate the virus by ultra centrifuging them (with PEG it the efficiency was down to 20%). I do the infection with the cells in suspension and it still sucks.
If you guys have any thoughts on how to improve this please share them with me. I am totally stuck!



#3 Radish

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Posted 26 January 2011 - 11:40 AM

Hi Clare

I tried other cells and they are fine, I can get up to 90 % efficiency, it is a ES cell thing apparently.
I can't use polybrene, it is extremely toxic to the cells, even at very low concentration (like 2 ug/ml).
I see a lot of people getting crazy efficiencies and I am not even close...
Tried less cells, doing it in suspension (after trypsinizing them), rotating them, you name it...

I think I need to find a decent virus titration protocol and play with the viral titers :(

Thank you for you reply Clare!

#4 Clare

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Posted 27 January 2011 - 12:55 AM

Hi Radish,

What kind of ES cells are you using?

I am growing rat ES cells and just 2 days ago infected them with a lenti using 8ug/ml polybrene and they look fine and happy.

I have the cells growing in 4 well plates (you know, the tiny little wells?) and left the cells in 200ul media + virus + polybrene for 3-4 hours only, then top up with fresh media (to 900ul).

Does this help?

Clare



Hi Clare

I tried other cells and they are fine, I can get up to 90 % efficiency, it is a ES cell thing apparently.
I can't use polybrene, it is extremely toxic to the cells, even at very low concentration (like 2 ug/ml).
I see a lot of people getting crazy efficiencies and I am not even close...
Tried less cells, doing it in suspension (after trypsinizing them), rotating them, you name it...

I think I need to find a decent virus titration protocol and play with the viral titers :(

Thank you for you reply Clare!



#5 Radish

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Posted 27 January 2011 - 01:41 PM

Hi Radish,

What kind of ES cells are you using?

I am growing rat ES cells and just 2 days ago infected them with a lenti using 8ug/ml polybrene and they look fine and happy.

I have the cells growing in 4 well plates (you know, the tiny little wells?) and left the cells in 200ul media + virus + polybrene for 3-4 hours only, then top up with fresh media (to 900ul).

Does this help?

Clare




Hi Clare

I tried other cells and they are fine, I can get up to 90 % efficiency, it is a ES cell thing apparently.
I can't use polybrene, it is extremely toxic to the cells, even at very low concentration (like 2 ug/ml).
I see a lot of people getting crazy efficiencies and I am not even close...
Tried less cells, doing it in suspension (after trypsinizing them), rotating them, you name it...

I think I need to find a decent virus titration protocol and play with the viral titers :(

Thank you for you reply Clare!



Hello Clare

Thank you so much for you reply.
So, it seems that you are using the Ivanova protocol for the infections...
I am really thinking to give it another go.
I am working with E14s, and I tend to see a lot of toxicity with polybrene (I do the transfection o/n)...
Maybe I should try to keep them in virus for less hours?
Tell me what you think.

Once again, thank you Clare


Best regards
Radish

#6 Clare

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Posted 28 January 2011 - 12:37 AM

Happy Friday!

I would try culturing them in virus + minimal media for 3-4 hours - that's the way the lenti lab routinely does it as my work, and it all seems fine :D

Clare



Hello Clare

Thank you so much for you reply.
So, it seems that you are using the Ivanova protocol for the infections...
I am really thinking to give it another go.
I am working with E14s, and I tend to see a lot of toxicity with polybrene (I do the transfection o/n)...
Maybe I should try to keep them in virus for less hours?
Tell me what you think.

Once again, thank you Clare


Best regards
Radish
[/quote]

#7 Radish

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Posted 31 January 2011 - 12:33 PM

Clare <3

You made me smile on Friday LOL, just didn't have time to reply.

3 to 4 hours is to much to have the cells in suspension, and to little to get the cells attached and take out the media+virus without loosing A LOT OF CELLS :(

don't know what to do :/

#8 Clare

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Posted 01 February 2011 - 12:38 AM

Hi Radish,

Do your cells grow on feeders normally? Mine do. So what I did was plate the cells onto fresh feeders one day, then the next day I put them in minimal media + virus for about 3 hours etc etc.

Clare

Clare <3

You made me smile on Friday LOL, just didn't have time to reply.

3 to 4 hours is to much to have the cells in suspension, and to little to get the cells attached and take out the media+virus without loosing A LOT OF CELLS :(

don't know what to do :/



#9 Radish

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Posted 01 February 2011 - 09:54 AM

No, I can't grow them on feeders (I use gelatin), I guess the only alternative I have is to use higher virus titer :(




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