Harvesting virus - (Aug/30/2006 )

Hi there

I've read a couple of protocols now, but still not sure what to do, and don't want to waste valuable time by doing it again and again.

The problem I'm facing is all protocols for lentiviral transfection, says that posttransfection harvest virus-containing supernatants 48-72 hours after. Now is that the cells that need harvesting or just collecting the media suspension that the cells were in? I'm taking it that it is normal harvesting of cells and then spinning it down to collect supernatant afterwards.

Please can someone shed some light on this?

Cheers
T
x

i think it depends on the cell type u are using

I'm using 239T cells, which is adhered to the bottom of the flask.

i'm not sure, but if u r going to spin down the (cells+media) and harvest just the supernatant , it would be the same if u only harvest only media supernatant and spin it down
i mean, in both cases you are going to end with the supernatant

in one protocol, i read that if u want the virus for in vivo application, it's necessary to get rid of cellular debries, otherwise it's optional





hope these protocols help u:

Thanks a lot for your help, I'll try it and hopefully it will work. Will let you know!!

Cheers

i'm not sure, but if u r going to spin down the (cells+media) and harvest just the supernatant , it would be the same if u only harvest only media supernatant and spin it down
i mean, in both cases you are going to end with the supernatant

in one protocol, i read that if u want the virus for in vivo application, it's necessary to get rid of cellular debries, otherwise it's optional





hope these protocols help u:

[attachment=1601:attachment]
[attachment=1599:attachment][attachment=1600:attachment]

i transfect all plasmids in the evening and change media nextday morning. Then 24hrs after media change, collect the supernatant and add more media for collecting in 24hrs.

I spin the supernatant and filter to remove any cell or debris.

I dont harvest cells.

I spin my supernatant as there r many dead cells and I want to get rid of them. Sometimes even after I spin and filter them, I still get some cell debris in my virus prep.

i didnt understand what u meant by "you'r working with plasmids, cells are important therefore"

To make virus, u need to transfect ur plasmids into the right cells. And for lentivirus, they r secreted into the media so it makes easy to simply harvest the supernatant.

My protocol: see atachment. Similar (if not identical) to Scolix's.