You are using the second generation lentivirus I suppose. We have used 3rd generation lentviruses to infect ESD3 cells with good success. What kind of selectable marker you are using? I used puromycin.

Briefly our protocol:

1) Seed cells at 1000 cells/mm2 in a 24 well plate. Allow the cells to adhere. For our cells it used to be 2 hrs.

2)Remove the media (which will essentially remove all the unattached cells).

3) We use 100X conc. Viruse (ultracentrifuged) 50µL virus+ 200µL media and 8µg/ml polybrene.

4) Allow infection to happen overnight. Next day remove the infection mix and add normal media and allow the cells to recover.

5) Once the cells look normal by morphology, then start selection by using desired amount of puromycine, some cell will die with 4µg/mL puromycine, some might need 8µg.

6) Select until all the cells in control die.

7) If this protocol does not work, may be there is not sufficient viral threshold to infect your cells, then you will have to optimise your viral production to get higher amount of virus. Share your virus production protocol, we can try to incorporate some changes in you protocol, so you can get high efficiency virus.

Regards,

Sandhanakrishnan

These are B lymphocytes, right? Retroviral infections of this cell type is extremely difficult.

Take a look at this thread:

https://www.researchgate.net/post/How_can_one_infect_human_B_cells_with_lenti_or_retroviruses

Hi Nicholas,

I agree with Daniel. I have had first hand experience with lentiviral transduction with primary B cells. They are extremely difficult to transduce. I have had best success with spinoculation followed by incubation at 32C instead of 37C since the virus is more stable at this temperature.

All the best with your experiment!

Thanks,

Siddha

Hi Nicholas,

Effectively this type of cells is very difficult to transduce, but we already have had positive results in this field (production by ultraC). I don't know your experimental context but you have to optimize several parameters:

- Lentiviral vectors with very high titers : 1E9 to 1E10TU/mL

- Adapted promoter (I advise PGK or EF1)

- Conditions of transduction : small volume and little number of cells

- You can add DEA-Dextrant in the medium (1µM final, 4-5H max) during the transduction step to enhance the entry of the vector in the cells

You can have a look here about protocols  of Lenti transduction according your type of cells

http://www.geg-tech.com/Knowledge/protocols/transduction-with-lenti-one

Personally, I don't like very much the polybrene, I think the results obtained are so different (sometimes enhances but sometimes decreases the efficiency) according the experimental context.

If you describe more your experimental context (promoter, titer of your vectors, protocol of transduction....), it will be easier to bring advices to optimize the transduction of your cells.

Best,

Nicolas

Thankyou all for your responses.

Nicolas in response to your questions:

I am not sure of the viral titre, but I am using a MOI of 50 compared to transfection of MEC-1 cells (a CLL cell line).

I have a vector with a CMV promoter and another with a B cell specific promoter (Emu-CD19 promoter).

At present I have been trying to transfect about 5x10^5 cells in a 500ul volume.

Thanks

Nick

As described through the link I put, I advise you to transduce cell in 24-well plate in a volume of 200ul with 10^2-10^4 cells.

About the promoter CMV is not a realy good promoter in your experimental context but Emu should be.

I advise you also to do a condition with DEAE-D

Let me know if you have other question or about the results of your next transductions!

Nico

Hi Nicolas,

I wonder have you get good result in this case? Can you share your experience with us. I am trying to transduce B cells too. 

Thanks

Hi Roghaiyeh,

Could you send me more information about your experiment?

You can send me that here

[email protected]

regards