adenovirus infection: 293FT Vs 293A cells - low titer virus despite high CPE! (Mar/14/2006 )
Hi,
I was wondering if anyone could help me with this. I recently infected adenovirus bearing a gene of our choice into 293FT cells. The CPE was very limited after 48 hrs and it took 5 days post infection for there to be complete CPE. At this point, I couldn't tell if the cells were simply dead coz they'd been in the same media for 5 days or whether the virus had actually been amplified and subsequently infected the remaining cells! I collected the virus anyhow and tested it and quite surprisingly, it showed that the gene of choice was expressed in cells infected with this virus. I did a Western Blot basically.
Then I tried another amplification of this virus using 293A cells. I had complete CPE within 48 hrs and I thought the virus would be high titer. Unfortunately, when I ran a western blot with cells infected with this new batch of virus, there was absolutely negligible amounts of protein expressed!
I'm confused now. I thought the 293A cells were better suited for adenovirus amplification coz the E1A and E1B genes are stably integrated into these cells and the CPE was ~95%. Did I make a mistake in waiting for complete CPE and lose the virus in the supernatant? Shld I collect the virus when the CPE is ~50-60%, please help..I'm quite new to this and will appreciate any advice.
Thanks a lot 
Hi
You should have waited for complete CPE before harvest, then the virus is at its maximum levels. I use 293 for Ad5 and mutants harvest and have found CPE to be approx 3-4 days typically.
BTW A293 are supposedly made by someone who took some 293 and serially and briefly trypsinised the cells to remove loosely adherent cells , thereby selecting for adherent cells, hence the "A" in front of the 293.
Cheers
Bob
You should have waited for complete CPE before harvest, then the virus is at its maximum levels. I use 293 for Ad5 and mutants harvest and have found CPE to be approx 3-4 days typically.
BTW A293 are supposedly made by someone who took some 293 and serially and briefly trypsinised the cells to remove loosely adherent cells , thereby selecting for adherent cells, hence the "A" in front of the 293.
Cheers
Bob
Hey Bob,
Thanks so much for your advice. I did wait for complete CPE before collecting both times. This is the protocol I followed:
1. Upon complete CPE, I collected the supn and the few cells adhered to the bottom of the dish.
2. Spun down the cells, 5K for 15-20 mins and discarded supn.
3. Resuspended the cell pellet in 5 mls of complete media, freeze-thawed 4X, spun again and aliquoted the supn that contained the virus.
I'm just confused about whether I lose a lot of virus in the supn following the protocol this way. Also, a former colleague had told me to collect the virus before complete CPE and I don't remember why! I'm in a new lab and am trying to troubleshoot this protocol.
Thanks for your help again.
Hi
Actually I should have mentioned that it is actually just before complete CPE that you should collect the cells, this is when the cells are not floating off, but look like they are about to, at this point the cells have packaged virus inside them which can be collected by centrifugation of the cells (gently, 250g for 10 minutes is fine) and then discard the supernatant which will contain lysed virus and cell debris. The Cell pellet then contains most of the virus and can be lysed to release it.
Cheers
bob