Here is the protocol for concentration (the second part of lenti production). You will not see the pellet if you have only virus. Usually you see the pellet of some cell debris and should get rid of the debris by additional centrifugation. You could titer a virus just by infection of HEK cultures with consecutive dilutions of a virus, if you have a way to detect your transgene (fluorescence or Ab staining). Otherwise you could do real-time or ELISA. Culture infection is more precise since there are always many damaged particles that do have viral RNA, but will not be able to infect a cell in vitro or in vivo. Real-time will detect damaged particles. And ELISA will detect also empty particles.

7) Transfer media from plates to 50 ml tubes. Spin down at 1100 rpm/200 g for 5 minutes. This step precipitates cells and big crap.

8) Filter supernatant through 0.45 um (or 0.22) syringe filters.

9) Transfer virus into centrifuge tubes. Cover each with parafilm.

10) Centrifuge the virus for 1.5 hrs at 25000 rpm (SW28 rotor of Beckman, ~120,000g), 4C.

11) In the hood. Pour off the supernatant. Turn the tubes upside down onto a paper-towel to drain for about 2-3 minutes. Remove excess medium from the sides of the tubes using paper-towel. The serum from the medium damages viral particles, so the tubes should be very dry, but not completely dry.

12) Dissolve virus. Add 100 ul of sterile D-PBS. If you want a more concentrated virus, you can go down to 50 ul.

13) Resuspend the pellet in D-PBS.

14) Transfer to 1.5ml eppi and centrifuge for 1 min at max speed on benchtop centrifuge. Carefully take the supernatant (pellet contains cell crap and serum factors). Aliquot and keep at –80C.

Hi Anil,

depending on the scale of your production, you should definitely see the lentiviral pellet as a yellowish, glassy spot at the bottom of the tube (using a swing-out rotor). In a fixed-angle rotor, the pellet will be dispersed along the side of the tube, which makes it impractical to collect in a small volume.

What type of rotor are you using at 15000 rpm, i.e. what is the g force (RCF value)? LV particles are ca 100 nm in diameter. To pellet these particles, we use ca 100000 x g for 2 h. In a Beckman SW28 ultracentrifuge rotor, this corresponds to ca 24000 rpm. Most likely, your g force at 15000 rpm is just not high enough to sediment the particles.

You don't have an ultracentrifuge at hand, an alternative method to concentrate the particles is ultrafiltration through a 300 kDa cut-off ultrafilter. 1000 kDa cut-off filters allow for faster concentration (with some loss of virus), but they are fragile (risk of leakage).

Again depending on your scale, you may need to process fairly large volumes of supernatant. Look for 50 ml-format ultrafiltration units containing a slanted membrane insert, for increased membrane surface and faster ultrafiltration. Make sure to pre-clear the supernatant by filtration though a syringe-fitted, 0.45 µ disk filter before ultrafiltration, to avoid clogging of the ultrafilter by debris.

There are several ways to titer Lentivirus, for instance:

1. Count genomic RNA copies by RT-qPCR (divided by 2 = number of particles containing RNA).

2. Quantify a viral matrix protein (e.g. p24) by ELISA

These methods give a physical titer, but say nothing about the infectivity of your particles, which is mediated by the viral envelope glycoprotein (pseudotype).

To determine a functional (infective) titer, use a cell line which is highly receptive for the pseudotype you are using (e.g. VSVg works well with HEK-293, or the packaging cell line 293T). Also, the internal promoter in your lentiviral construct must be active in the indicator cell line. Transduce the cells at ca. 30% confluence, e.g. in a 48-well format, with serial dilutions of your viral stock, down to clonal dilutions (10^6 to 10^8). Leave the cells to grow for ca. 3 doubling times (ca. 3 days to confluence, for 293-type cells), then fix and immunostain the cells for vector-expressed protein (conveniently, the fluorescent reporter co-expressed with your gene of interest).

Using a regular epifluorescence microscope, in samples transduced with clonal dilutions you will see individual clones containing 2-8 immunopositive cells each. From the number of clones per sample, the dilution factor, and the volumes used, you can calculate the functional titer (=transducing units/ml in your original viral stock).

This method relies on immunodetection of gene expression by one single copy of integrated vector per cell. Not every integrated copy will express protein levels above detection limit, so the number you calculate will underestimate the "real" functional titer. Anyhow, you may compare one viral prep with another, using the same titration method. Good concentrated stocks should have ca. n x 10^10 to 10^11 tu/ml.

Hope this helps,

Alex

Hello Anil,

no, you won´t see a pellet and the centrifugation is already the easy way to concentrate lentiparticles. Which transgene did you package into the particles? GFP? Do you have a microscope? Then the titer can be calculated from the amount of green cells after 24h. (%positive cells x seeded cells/µl vector stock solution)

All the best,

Irene

Thanks a lot to you all for giving me suggestions. I will go through practically doing them one by one.

Best,

Anil

Hi Alexandra,

I am a student of Dr. Anil Gaikwad. I am trying to prepare and concentrate lentiviral particle but failed to titre it. For titration you have suggested to count genomic RNA copies using RT-PCR. Can you suggest me what different types of primers you have used for RT-PCR.

Waiting for your reply

Thanks

Hi Anil,

sorry for delay, holidays...

Practically, pick primers directed to regions shared by most regular lentiviral plasmids: E.g. the 5'-region between U5 and the 5'-end of the gag gene, and/or the WPRE. This way you may use the same primers for all your constructs.  Find some good primer suggestions in attached paper.

Good luck! :)

Thanks Alexandra for the help!

Best,

A

Gireedhar, I use SW28 to spin vector. Did you fill your tubes? SW28 tubes are 40ml open top tubes (Beckman ultraclear) and should be filled with 36-38 ml or they collapse. Top up with PBS if you don't have enough sample to spin. If leaky you mean a little bit was under the tube when you removed, it is more likely your hands were wobbly than your tubes were leaky. It is easy to forget you have an open top tube filled almost to the brim when carrying the buckets to the rotor and dropping the rotor down into the centrifuge.

Hello there,

I got a couple of pretty trivial questions.

1) How do you guys maintain sterility with open top tubes?

2) Re-suspension: how long will take the viral pellet to be fully re-suspended?

Thanks a bunch.

Hi Davide,

for sterility I only open the tubes in the cell culture cabinet using etoh sprayed gloves, fill them and put them in the buckets and do up the lid all inside the hood. then the closed buckets go to the ultrafuge, once spun they come back into the hood before opening. The bucket lids and top edges should be wiped down with ethanol. I know other peoples strategy is to spray etoh on parafilm then use that to seal over the tubes before taking them out of the hood. Resuspension, I've been told it should be soaked for around an hour on ice between actively pipetting but don't know how true that is; personally I pipette up and down for about a minute per tube - I am happy that my titres are among the highest reported using my vector.

Thanks a bunch, Nathan.

Your strategy makes a lot of sense to me.

Thank you again.

We also tried concentrating our LV with 16.000 g for 3,5 hours and it did not work. As you said this might not be sufficient, but it seems to work for other viruses at our institute.. how is that possible?

Or is this maybe just the cell debris?

Amicon and Centricon centrifugation membranes can boost titer to 100-fold without more than 10% loss of the virus.  Check out Millipore's website for the 3kDa membranes depending on your scale.

Although, this is an old post, but for new viewers these products from TaKara can be very handy for good research and Good science. Hope this will save you lot of time and better results.

http://www.clontech.com/NL/Products/Viral_Transduction/Lentiviral_Transduction_Tools/Lentivirus_Titration/Lentivirus_Titration_Overview?sitex=10140:22372:US

http://www.clontech.com/NL/Products/Viral_Transduction/Lentiviral_Transduction_Tools/Lentivirus_Concentration/Lenti-X_Concentrator?sitex=10140:22372:US

Many thanks to everyone that have contributed to this post. I have really learned a great deal on how to improve my Lentivirus production.

A couple years ago someone gave me a protocol to filter concentrate with tabletop centrifuge. We filter soup through 45um filters to prevent contamination of target cells with 293T cells. You can place up to 15ml into an Amicon filter concentrator (100,000 MW cutoff will filter out the albumin) and concentrate to 200-300ul by centrifuging at 3000xg for 15minutes. It is a workaround if you don't have access to an ultracentrifuge and takes very little time.

Can we use fixed-angle rotor to centrifuge lenti preps? Many thanks.

Back during my graduate days, I used this approach:

https://www.nature.com/articles/srep13875

Worked just as well as advertised in my hands with cheap, easy to make reagents and equipment. But I have to say, I still greatly prefer rAAV over the lenti approach.

Hey Gerard, I am wondering what kind of tube you used for the lenti-virus concentration work@ Gerard Joey Broussard

Hi Mingxiang,

I used standard 50 mL conical with an angled rotor. Best of luck with your experiments.

- Joey

Thanks so much Joey@ Gerard Joey Broussard

Concentrating lentivirus particles and titrating them without a kit can be achieved using standard laboratory techniques. Here’s a guide to both processes: ### Concentrating Lentivirus Particles 1. **Ultracentrifugation**: - This is a common method for concentrating lentivirus. - Spin the lentiviral supernatant at a high speed (typically around 25,000 rpm) for a few hours at 4°C using an ultracentrifuge. - After centrifugation, discard the supernatant and resuspend the viral pellet in a smaller volume of appropriate buffer (like PBS) to achieve the desired concentration. 2. **Ultrafiltration**: - This method uses a membrane filter to concentrate the virus. - Pass the lentiviral supernatant through a centrifugal filter unit with a suitable molecular weight cut-off (usually 100 kDa). - Centrifuge according to the manufacturer’s instructions, which will retain the virus on the membrane while the smaller particles pass through. - The virus can then be resuspended in a smaller volume. 3. **PEG Precipitation**: - This involves adding polyethylene glycol (PEG) to the lentiviral supernatant to precipitate the virus. - Incubate the mixture overnight at 4°C, then centrifuge to pellet the virus. - Resuspend the pellet in a smaller volume. ### Titrating Lentiviral Particles Without a Kit 1. **p24 Antigen ELISA**: - While this method typically uses a kit, it can be performed without one if the necessary reagents are prepared in-house. - The p24 protein is a viral capsid protein, and its concentration can be correlated with the number of viral particles. 2. **Functional Titering**: - Transduce a known number of cells with serial dilutions of the virus. - After an appropriate incubation period, measure the transduction efficiency. This can be done through fluorescence microscopy if the virus expresses a fluorescent protein, or by flow cytometry for cell surface markers. - Calculate the functional titer based on the percentage of transduced cells and the dilution factor. 3. **Reverse Transcriptase (RT) Activity Assay**: - This assay measures the activity of the reverse transcriptase enzyme present in the viral preparation. - It requires the preparation of an RT reaction mixture and a substrate, followed by incubation and measurement of the reaction product. - This method provides an indirect measure of viral particle concentration. ### Considerations - **Safety**: Lentiviruses are biohazardous, so all procedures should be performed in a biosafety cabinet using appropriate safety protocols. - **Efficiency**: The concentration and titering methods have varying degrees of efficiency and accuracy. Ultracentrifugation is generally more effective for concentration but is time-consuming and requires specialized equipment. - **Standardization**: It's important to standardize the method of titration for consistent results, especially when comparing different batches of virus. These methods provide feasible ways to concentrate and titrate lentivirus particles in a laboratory setting without the need for specialized kits. However, the choice of method will depend on the available resources, the required level of precision, and specific experimental needs.

l This list of protocols might help us better address the issue.