Hi, We have observed this virus "going off" within 2 months as well. ...any idea why this happens?
Chitra On 31 March 2011 18:07, Nathaniel Clark <nathanielcl...@gmail.com> wrote: > 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 > >> > >> > > >
