In article <2hdfk0$4jm at mserv1.dl.ac.uk>, <unknown at dl.ac.uk> wrote:
>Hello, looking for any suggestions. How would one go about determining the
>titer of an animal virus in mammalian cells grown in suspension? These cells
>(lymphoblastoid lines) do not want to adhere to most things, and I am concerned
>with being as quantitative as possible. I am not interested in looking for
>replication (ie plaquing units), but would like to know how many cells express
>these viral products (I have antibodies). I have considered immunoflourescent
>microscopy but since these cells do not like to sit down, I do not see the
>feasiblity of this approach. I am also looking into flow cytometry, but am
>concerned with sensitivity. Also, several of the proteins I am interested in
>are intracellular. Please post any response or mail me directly. I am...
>much obliged!
>>Brett Lindenbach
>Washington University - St. Louis
>brett at borcim.wustl.edu
Brett,
we do a lot of IF of suspension cells. We spin them down, wash them (all
in eppendorf tube) and remove washing sol (PBS) by suction, but leave
about 30 ul of PBS (depends on amount of cells or size of pellet) and
resuspend cells in this small vol of PBS. Thus we get thick solution
of cells, which we apply on welled microscopic slide, just the amount we
have enough cells within each circle. Then we leave it to dry out, and
then fix by aceton. The cells remain stuck to the glass and we can
apply imm. reagents in two steps. Then you can count amount of IF+ and IF-
cells.
You can do this by using more sophisticated apparatus - cytospin centrifuge.
Otherwise, you can measure the "titer" of virus by finding out in what
time you get certain level of CPE by infecting your cells with certain
dilution of your virus prep.
If you want to know more, e-mail me.
Pavel
Pavel Sova
Molecular Virology Laboratory
Columbia University
New York
