So, I have transfected 293HEK cells with AAV-GFP vector and after purification, I ended up with 50ul of purified virus (or so I hope). What would be the best method to determine my titer with the amount of virus that I have.
Thanks.
I haven't done AAV but this is what I do for lentivirus. Though lenti integrates into the genome and AAV doesn't, I think this method should still translate...
Transduce a well of cells (293T or HeLa or something) with an aliquot of the virus. I usually do a curve covering a couple of log orders and in duplicate. After 48 hours you can measure the number of GFP+ cells by flow cytometry (or some other counter if you have one) and use Poisson statistics to deduce how many infectious units (IU) are present per unit volume. You can do it using something like this:
proportion untransduced (GFP-) cells = e-λ, where λ is the IU/cell. Rearranging for λ:
λ = -ln(neg. proportion)
Once you have λ, you can work out your IU/uL based on how many uL of virus was added and how many cells it was titered over.
OK thanks!
Do you know whether I can bring up my stock volume with DMEM to have a higher working volume (because all I have is 50ul)? Will that affect the assay?
You could...or alternatively take an aliquot of the concentrated stock and dilute it down. It's hard to know what starting volumes are best without having an idea of how many units might be there. Maybe for a first test you could use 1uL of stock, 1uL of 10X diluted, and 1uL of 100X?
The measured titer also depends on the method used...
This would mean that as long as everything remains the same (host strain, growth cycle status, medium etc.), you will get reasonably consistent results. Fortunately, AAV seems to tolerate freeze-thaw better but it is always a good idea to aliquot stocks in siliconized sterile tubes in portions that are sufficient for a single day's experiment.
Best,
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