adeno virus infection - (Jun/30/2009 )

Hi

I did infection with adno virus and after 1 hour of infection (2 ml of medium without serum),
almost 50% of the cells were floating. I know it isnt soppose to be like that.
do you think that the infection was too long? high titer of the virus? or maybe the cells werent good enogh? although they look very good to me before the infection.

I appreciate any kind of help

Shani

What sort of cells?

Adenovirus infection usually takes at least 4 hours to produce any significant virus output so I suspect that your result is due to some other factor such as the lack of serum, or a change in pH, infecting too close to seeding etc...

What is your virus titre?

bob1 on Jun 30 2009, 04:15 PM said:

the cell are hek293 with E1a-E1b.

I dont know what the titer. im try to do amplification first and then check titer.

Uaually in our lab, we do the infection for 1 hour with medium without serum, and after 1 hour we add 10 ml of medium with serum.
maybe this fact hurt the cells?

293 should be fine without serum for a few hours, but are known for not being very strongly attached to surfaces. You could leave the virus on for a bit longer (2-3 hours) or coat your plates/flasks with gelatin to help the cells attach. Otherwise you could try doing a titration of the effect on 293 plated out in a 96 well plate. You will need to seed the cells wait 24 hours, then add different dilutions of your virus prep, and see if there are still lots of cells floating with the higher dilutions.

Thank you Bob!

I coated my flask with pll and it was much better.

I have a funny question. when i freeze my virus (in the freeze\thaw stage), should I freeze it untill all the liquid turn to white? or until the liquid is frozen?

thanks again

shani

Freezing until the liquid is frozen is all that is required, it is the formation of ice crystals in the cells that disrupts the cell membranes, releasing the virus.