We do adherent if we have a small volume of low titer virus, but as soon as we have a decent titer we will start with a 30 ml suspension flask. Normally I add something like 0.5 ml, which is probably more then I need. We only use serum free media right now. As I mentioned, if you need to boost the titer of a low titer stock, we plate a t25 flask with Sf9s, take off the media, and incubate it in 1 ml of pure virus (or two-fold diluted) for an hour or so, then remove (and save) that virus stock and give the cells fresh media.
For fold dilution, just do a rough calculation of titer. Once you are at a high titer assume you have 10^8-10^9 pfu/ml, so you can estimate the ballpark volume to infect a given number of cells at an MOI of 1. >From early stocks you might have a titer of 10^6-10^7 pfu/ml. If you do an amplification that doesn't work, decide if you were to high (MOI=>10 not great for amplifying) or too low (MOI=<0.1) and try again. Better yet, just set up 3 amplifications at the same time, whichever worked the best, use for another round of amplification, or for infections. For duration, we do 3 days amplification (post-infection), but sometimes we let them go a few days longer, which allows a secondary infection of the cells that weren't infected by the viral innoculum. There doesn't seem to be any harm to letting infections go longer; at some point all the cells die, but since the virus is pretty stable, it's no problem. We have a CEDEX cell counter which tells you average cell diameter, which is a great read out for infection. Also looking and density and viability versus an uninfected control will tell you if the infection worked. Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst On Thu, Mar 31, 2011 at 12:36 PM, Katya Heldwein <katya.heldw...@gmail.com> wrote: > A related question: how do most people amplify their baculovirus > stocks? Adherent cultures vs suspension? Fold dilution at each stage > (P1 to P2, P2 to P3)? Duration of each amplification stage? > > We have some viral stocks that "go off" rather quickly (1-2 months) > despite being stored with FBS in a cool, dark place. > > > Katya > > > On Thu, Mar 31, 2011 at 10:23 AM, Brad Bennett <bradbennet...@gmail.com> > wrote: >> Though I'm not meaning to turn this into a plaque assay burn session, for >> many reasons we have also abandoned it and instead titer our baculovirus >> stocks (P3 and P4) using flow cytometry. We use an antibody against viral >> gp64 that has been labeled with PE and stain infected cells in 96 well >> plates with the PE-antibody. We then measure PE fluorescence on a flow >> cytometer (you can also do cell counts, viability determinations, etc.). We >> equate 1 fluorescent event as 1 infected cell. Since we know how many cells >> have been plated in each well, we can determine the percentage infected in >> each well. We calculate a "non-normalized" titer from this data alone or we >> compare this data to a standardized virus and determine a normalized titer >> using a standard curve. From infection to having a titer in hand takes about >> 24 hours. Of course, the potential bottleneck is access to a flow cytometer! >> I can give more experimental details off-board. >> I should say that for getting an idea of relative titers and to test protein >> expression on a small scale, we also do the "effective titer" tests as >> suggested by Nat, with cell morphology and immunoblots as our read-out of >> virus potency and recombinant protein expression, respectively. No doubt, >> this will get you a long way but at some point, I argue, you need to >> determine an actual, absolute titer for duplication of results, >> troubleshooting, monitoring virus health over time, publications, etc. >> >> HTH- >> Brad >> >> On Thu, Mar 31, 2011 at 9:23 AM, Bertrand, Jay Aaron [Nervianoms] >> <jay.bertr...@nervianoms.com> wrote: >>> >>> 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 >> >> >
