4 replies to this topic

[fguilhem]

hi,
i'm using 293 PACK cells (also called Phoenix cells) for infecting 293 Cells. Is it neccessary for a good infection ( and therefore good retroviruses production) that 293pack cells divide them continually?
Do someone know tips for achieving good infection (apart the fact that target cells must divide them) ?

Many thanks.
Fred

Edited by fguilhem, 09 February 2005 - 11:46 AM.

[laurequillo]

Well, but you need is to introduce your plasmids, and then the cells will produce the virus. You just have to take the supernatant (I think it is 24-48 after transfection) and do a titration on 293 just to see which volume of supernatant is the optimal for your goal (silecing or whaetever)

I dont know if that was your question

[LostintheLab]

If you got these phoenix cells comercially they should have protocols for transfection with ideal conditions- its probably wise to follow their recommendations.
If not, then....
I haven't used those particular 293 cells, but I have used other 293 for viral production and low passage number for the cells is important for transfection efficiency- the younger the better. Also don't let your cells get too confluent, around 60-70% is optimum for tranfection of these cells, which can be tricky when they grow quite fast.

In other cell lines lower cell confluence is recommended for tranfection efficiency. What I tend to do is set up a range of cell confluence or vector concentrations to find the best- it doesn't have to be a big range, one or 2 different cell densities or vector concentrations.

[warsel]

Just to add another opinion:



I use 293T cells for packaging and usually transfect in a 6-Well with CaPO4 (at 30-40% confluence) over night, change the medium (1-2mL) the next day in the morning, harvest virus the day after (you can harvest twice ~8h apart and the following day as well if the cells are still ok).

Using a 3-plasmid lenti system and pLKO.1 vectors this usually gives a >90% infection efficiency judged by puromycin resistance at 1:5 dilution (virus:medium) in our hands.

[miBunny]

Some tips!

Never let the packaging cells overgrow!

Monitor for mycoplasm infection

The day before you replate the cells for the transfection, remove half the media and replace it with new.  This would keep the cells "revved up" and helped increase the viral titer.

I would plate the cells in opti-mem with 5% FBS for the transfection.  The next day, I would remove the Opti-mem and replace it with regular DMEM-10 for the viral production.

Optimize your transfection efficiencies to get 90-100% transfection.  I would make multiple batches of 2X HBSS at slightly different pHs and use the best one for my transfections.