Every 24 hrs should be best based on the protocols I've read. This will clear the supernatant and give the viruses increased opportunity to produce.  Here is one example protocol, http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/shrna/learning-center/lentiviral-packaging.html.

I agree with you, but my concern is that if the viruses are left in the supernatant too long, would they be able to infected the 293T cells so you end up with less viruses.

Best!

Haitao

I don't understand your last question, why would the virus be left in the supernatant too long? You collect after 24h and either use the virus or freeze it.

I collect virus after 48 hours and get good results afterwards.

Do you know if the produced lentiviruses in the supernatant can infect the rest of untransfected 293T cells in the wells/dishes (assuming the initial transfection rate is not 100%). If they do, are you going to lose more viruses when you collect once by 48hrs rather than collect twice every 24 hrs?  One more question: can you still get some viruses (may be at lower titer) at 72 hrs?    Thank you all.

From my experience, you can collect every 24 hours for anywhere up to 5-6 days after the initial transfection, but the yield will generally tend to fall towards the end where it is not worth the time or effort to collect it.  Ergo, you can still get viable infectious vector at 72 hours, but it really depends on how good your peak vector production is.  If it is in excess of 10e7 GFP-positive cells after 36-48 hours, then likely your 72 hour titers will be in the low to mid 10e6 vector range.  If it is lower than this, and depending on what you plan to use the vector for, then you need to decide if it is worth the time, effort and supplies to collect it.   Alternatively, you can always collect as many days as you want, and then concentrate the vector by ultracentrifugation.  This could effectively increase your vector concentration.

Thank you all for your advice! 

I understand your question about the lentivirus simply turning around and infecting the producer cells - I think that without an agent like polybrene this would only happen to a small degree. You could always test the theory by adding polybrene to the producer cells and seeing if your titre is reduced!

Anyway, I believe that lentivirus only has a half-life of 8-9 hours at 37 degrees, which is possibly why the supernatant is collected every 24 hours.

In my experience, 48 hours is the best choice if you are using calcium phosphate transfection, as this is the time where the expression levels are in the highest point. Also, you can collect a second supernatant 24 hours after the first one; you'll get less titer in this second collection, but more than enough for in vitro purposes. After this moment, there is no point in trying to get more lentiviruses. As I work mainly in vivo I only collect the supernatants after 48 hours, where the titers are higher.

I collect virus after 48  and 72 hrs,  and some cell lines with low transfect efficiency ,you can tranfect  twice

As comment to Jill Muhlings statement, that without polybrene no viral infection will happen: thats not true, depending on your cell line, you can get very high transduction efficiencies with just adding your undiluted virus.

Just finished my first lentiviral prep using GFP control vector from Packaging mix kit from GE Dharmacon.

(1) Transfection 293T for 12hrs, and then changed the medium to low serum (5%FBS)  DMEM; (2) collected supernatant at 24 (2ml), 48 (2ml), and 72 (2ml) hrs; (3) finally, collected the packaging cells together with 2ml of supernatant at 120hrs.

For the last collection (at 120hrs), I frozen down the cells/supernatant at -80C and thaw it after two days and spin down the debris at 2000rpm/5min at 4C.

Using the supernatant from the last collection, I did a simple titration with 50ul, 100ul and 200ul supernatant to infect 293T in 6 well plates. After two days, I can see more than 30% GFP positive cells with 200ul supernatant. 

The conclusion is that you can still collect viruses from supernatant at least up to 120hrs.

@Sophie yes exactly - depending on the cell line. In 293Ts transuction is fairly low without polybrene and that's why infection of producer cells isn't a big problem.

Hi Hai,

The best time to collect virus is just once at 48hrs.  You can technically collect virus at 24, 48, and 72hrs, however the virus is more dilute when you do this.  If you collect once at 48hrs, you will be collecting the virus you would of gotten at 24 hrs and 48hrs but in 2ml instead of 4ml.  I wouldn't go to 72hrs because the cells will be very confulent and you will not gain much in titer.

Good Luck,

Karen

48hrs post-transfection work better. If you add a new medium to collect again later; then expect a slim yield.

There is a Lentivirus Webinar coming up that may able to help address your concern and any additional questions.

Title: Knockdown, Knockout, Validate: Lentivirus delivers payload in vitro and in vivo Date: Wednesday, May 16, 2018 Duration: 60 minutes Times: 10:00 AM (CST) Speaker: @Christy Hoffmann

What you will learn: Lentivirus biosafety features and best handling practices Flexible customization options to fit your needs Techniques to incorporate lentivirus into your gene studies Insight from a leading Lenti manufacturing expert

Register here: https://www.sigmaaldrich.com/life-science/learning-center/customer-education/lentivirus.html