Hi

First, the more  differentiated cells get the more difficult they get to be transduced.

Second, you need to concentrate your virus in the supernatant. here is our way:

we concentrate virus in supernatant from each day after transfection using Amicon100 column (which are characterized for protein isolation but it works for lenti virus plenti good)  by centrifugation at 3500 RPM 4 degree

concentrate the supernatant from 10 cm dish to the final volume of  250 ul.

this has enough virus to transduce the cells in a 24-well plate. before adding viruses, remove the same volume of media  of target cells and slowly replace it with concentrated media containing virus while you are carefully add it to all over the well not just in one place of the well.

increasing polybrene might just kill your target cells.

Try not to seed too many target cells for transduction and keep it in mind not to increase too much of the volume of media containing viruses on the cells.

Replace the virus containing media with fresh media after 24 hours. if everything done as above, you should see the transduced cells at last after 72 hours after transduction, if you dont, you probably wont see them at all after that.

Have a good transduction!

Mokhlesim Seyed reza

Mostafa

Hi

First, the more  differentiated cells get the more difficult they get to be transduced.

Second, you need to concentrate your virus in the supernatant. here is our way:

we concentrate virus in supernatant from each day after transfection using Amicon100 column (which are characterized for protein isolation but it works for lenti virus plenti good)  by centrifugation at 3500 RPM 4 degree

concentrate the supernatant from 10 cm dish to the final volume of  250 ul.

this has enough virus to transduce the cells in a 24-well plate. before adding viruses, remove the same volume of media  of target cells and slowly replace it with concentrated media containing virus while you are carefully add it to all over the well not just in one place of the well.

increasing polybrene might just kill your target cells.

Try not to seed too many target cells for transduction and keep it in mind not to increase too much of the volume of media containing viruses on the cells.

Replace the virus containing media with fresh media after 24 hours. if everything done as above, you should see the transduced cells at last after 72 hours after transduction, if you dont, you probably wont see them at all after that.

Have a good transduction!

Mokhlesim Seyed reza

Mostafa

HI Syed,

we propose two types of proven and published solutions for increasing transduction efficiency;

1- Magnetofection which is a magnetic nanoparticles based technology that concentrate and improve your lentivirus infectivity

http://www.ozbiosciences.com/content/13-magnetofection

http://www.ozbiosciences.com/lentivirus-retrovirus/68-viromag-rl-magnetofection-reagent-viral-transduction.html

2- A chemical based booster much safer and gentle for the cells than Polybrene

http://www.ozbiosciences.com/transduction-infection/75-lentiblast-transduction-infection-lentivirus-booster.html

I hope this can help

Best Regards

Dont use too much polybrene as it is toxic for the cells. I used about 2 million particals when i transfected my stem cells. Also do you have a lenti-virus with some sort of antibiotic selection? that will help you a lot getting rid of the non transduced cells. Lastly it none work try to spin down the cells add the polybrene and virus to the tube for 2-4 hours and then re seed them. As i was troublshooting my transduction this method was brought to my attention. Good luck

Dear All, 

Many thanks for your kind suggestions. Let me make me points to you all:

MOSTAFA: For me latest transfection, I already concentrated the virus. I started with 10cm dish with 10ml, and first supernatant collection was at 48 hrs, next at 72(48+24). First harvest was approx 8 ml and then I concentrated it with Clone tech lenticonc. kit finally to that 8 ml (200ul in PBS). and then I first checked it in HEK293FT cells (as a control expt) with two conc. of 60ul & 20ul of the viral pellet dissolved, and within 24hrs I have a great result I believe it is almost 80% transduce (pictures attached both color & DCI image), now I am waiting for the NPCs to grow so that I can start next step with NPCs. 

Do you work on NSC/NPCs if yes then how much of your 250ul viral pellet you add per well in 24hrs well? and normally what is your confluency at the time of transduction, and also your seeding density please. Many thanks 

In this case (293) I started with 1,00,000 cells and it was about 75% confluent.

OLIVIER: At the very first I went to your site for lentiblast but since there were no related citation for the product with NSCs, and thus I dropped the plan, even though I checked it on pubmed but unfortunately unable to find the same. Do you have an experience of suggested product with NSCs transduction? Or if you know someone who has used the product for NSCs/NPCs, please let me know. Many thanks 

HADI: Thanks for the article I will look on.

LUAI: For the 293 cells(picture attached) I have used 2ug/ml polybrene, but no spin, but for my NPCs I will do the spin at 2000 rpm for 90 mints (as others to have suggested me to spin).  Infact I am working on CRISPR virus which has pyro selection marker, so If with the control mCHerry NPCs transductiion works fine then of course I have to select the NPCs with pyro selection. 

At what speed do you spin your polybrene and virus, and is it for 2-4 hrs? Many Thanks

Please find the attachments. Coloured fig. in pair with bright field images.

Pic No: 1 & 4 are of 20 ul virus pellet used ( mCherry & DCI)

Pic No: 2 & 3 of 60 ul virus pellet used (mCherry & DCI)

Many thanks to All of you.

Best 

Syed 

you spin down the cells and then after you have the pellet get rid of the sup and add the polybrene and virus (you dont spin down the virus and polybrene) let it incubate for 2-4 hours and then re-seed the cells

Ok, Many thanks I got it but spin at what RPM and for how long? Many thanks,

Syed

Glad to hear things are good with you.

the day before transduction, seed 50,000 cells per well of a 24-well plate. since you need to wait for  72 hours to see if the transduction has worked, they wont get too confluent till then.

you would get better result by harvesting 24 hours and especially 48 hours virus . you may never need 72 hour virus. if you have a good transfection and adjusted account of lentiviral plasmids, 24 and 48 hour are enough. 72 hours is less virus producing one.

if you would not harvest virus on 24 after transfection it may transduce the transfected cells which wastes your virus particles.

I have tried polybren before but  it never changed the result for me as I was able to transduce many cells with out polybren.

Best of lucks

Mostafa

800-1500 rpm for 10 minutes 

Manu Thanks Mostafa & Luai :)

Hi Syed,

If you want we can send a free sample of Lentiblast since this is a rather new product, we still do not have publications. You can contact us directly at [email protected] with all you information.

Best Regards,

Olivier

No problem

Dear Mostafa, 

As intimated earlier I did the mCherry control experiment in 293 cells to seethe efficacy of the virus I made, and concentrated and today is the Day 3rd but I think approx 90% of the cells are positively transduce. In this case I didn't centrifuge the 293 cell after viral complex addition. Today i am going to start NPCC control study to see the mCherry transduction, but thinking off to do a additional centrifugation step at 2000 rpm  for 90 mints. 

Please find the Day3, 293 cells cherry lento transduce pics. 

Many thanks,

Syed

Dear Olivier,

Many thanks for your kind reply. I would be more than happy to have a sample run of the Lentiblast and will let you know soon about the results. Will write to technical department about the same today. Many thanks for your offer, Syed

Dear Sysed

what vectors do you use? could you give me their commercial name?

and also do they have antibiotic marker?

I ahave psPAX2 (packaging) & VSVG (envelop) - Picture Attached, although both are a gift from the neighbouring lab.Infact mCherry is also shared from a nearby lab but unfortunately I don't have map of the same. And yes my CrISPR has a puro resistance marker, on what yet I have not started. I have an another packaging too pCMV-dR8.2 dvpr (packaging) but for the same I don't have map too. But both the packaging is working fine. You can go for anyone from Addgene, as these were originally purchased from Addgene.

I hope this will be helpful.

Best,

Infact I have asked for the map of the next packaging pCMV-dR8.2 dvpr (packaging) , if I get by monday then I will let you know. But for mCherry even that fellow has got it from someone else, so we have no information regarding the same.

Best,

Syed

That s ok, Thank you very much for kind answer. If there is no changes or digestions on the vector, map could be used from addgene. Thanks again.

Wish you luck in your research

Mostafa