I checked with someone in our protein production group and got the following response:
We also stopped doing the virus titration with the plaque assay and instead are performing expression test with different concentration of virus from the 3rd amplification. But for some viruses we still have doubts concerning the amplification success, so we are now evaluating a new technology using qPCR with the following kit (http://oetltd.com/products/category/baculoquant/). So you might have a look and see if it could be useful for your group. We would also be curious to hear if anyone else has experience with this approach. I hope this helps. Jay -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nathaniel Clark Sent: Wednesday, March 30, 2011 11:38 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] titering baculovirus ? 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? This message has been scanned for malware by Websense. www.websense.com
