8 replies to this topic

[Radish]

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!

[Clare]

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

Radish, on 25 January 2011 - 10:54 AM, said:

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!

[Radish]

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!

[Clare]

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

Radish, on 26 January 2011 - 11:40 AM, said:

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!

[Radish]

Clare, on 27 January 2011 - 12:55 AM, said:

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

Radish, on 26 January 2011 - 11:40 AM, said:

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

[Clare]

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  

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]

[Radish]

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 :/

[Clare]

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

Radish, on 31 January 2011 - 12:33 PM, said:

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 :/

[Radish]

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