We don't have a problem getting them to stick to the plates in serum-free media, or in 5% FBS media. The more challenging part is getting the plating density just right, too low and the plaques are too big, to high and they are too small. Or if the cells dry out, or if your agarose overlay is too hot, etc...
However, we have actually stopped titering all together. We find early stocks (from co-transfection, or plaque purification) are 'low', but after ~2 rounds of amplification in adherent culture of the 'low' titer stock(using a large volume of low-titer virus in a t25 flask), we can add ~0.5 ml to a 30-100 ml shake flask of Sf9's (serum free media) and get a high titer stock( ie. >10^8 pfu/ml). From there we amplify with ~ 10 mls of virus in a 1 L shaker culture, and we have our large volume high-titer stock. Sometimes we will incubate the cells in pure virus stock in a t25 flask for 1 hour, take the virus off, and add fresh media, as a way to rescue low-titer stocks. If you are just trying to titer (not plaque-purify), you can just take 10 fold dilutions of your virus, and do several small scale infections in 6 well plates, 10 ml shaker cultures, whatever you prefer. At the lowest virus concentration where you see a synchronous infection (judged by protein expression levels, or cell-diameter if you have a cell counter, or by viewing with a trained eye), you call that an MOI=1. From there you know the number of cells in the plate, and the volume of virus you added, so you can calculate an effective titer. Plaque assays are really difficult and slow, and if you are just trying to make protein, an effective titer is fine, the absolute number isn't that helpful, Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst On Wed, Mar 30, 2011 at 5:14 PM, Gloria Borgstahl <gborgst...@gmail.com> wrote: > Hi Guys, > we are learning to work with Sf9 cells and Carol in my lab wanted me to ask > you the following question. Many thanks for any help, G > > I need to titer a baculovirus stock in my suspension-adapted Sf9 cells. I > know that these can be encouraged to attach better to tissue culture plastic > if they have added FBS (about 10%), but am not sure that they will not be > migrating and hiding plaques. Does anyone have suggestions about how to > keep them more firmly anchored during the baculovirus titration, or about > another cell line that we could use instead?
