I had tried shRNA Lentiviral particles from Santa Cruz (sc-108083) and siRNA from Life technologies (Lipofectamine RNAiMax Procedure) but failed in both cases. I really appreciate your advice as soon as possible.
I did not work with these cells. However, with T cell lines, we have rather good transfection rates using Nucleofection technology from Lonza (and also NEON from Life). So if you have access to such a device, it would be worth a try. (Also it is strange the Lentivira do not work.... did you test them on other (e.g. HEK) cells?)
I would suggest to have a try with Viromer reagents, they give very good results for suspension cells. You can have a look to relevant data for immune cells on the following link https://www.ssl-id.de/www.lipocalyx.de/immune-cell-transfection/?force_sid=2s46gvn62mqvp05e1csoqi4a12
Have you tried changing the multiplicity of infection (MOI) of lentivirus to cells? You may have to go with a high MOI depending on your cell line. In some cell lines you need to use 10:1, 20:1, 40:1 virus to cells to get good transduction efficiency. Also, are you using polybrene during the transduction? The concentration of polybrene is somewhat cells line dependent.
Thanks everyone for your suggestions.
Christian Gabriel: I have not tested HEK cells
Tom Masi: I used polybrene at 5ug/ml as per protocol and I used 25,000 virus particle into 500,000 cells in 2 ml media
I believe your lentiviral transduction failed because the amount of virus you used was 20 times LESS than your cells. You should be using an equal amount or an excess of virus particles with your cells. The amount of virus particles needed to get good transduction efficiency is cell line dependent. You should check the literature to see if anyone has used lentivirus to transduce your cell type and use the MOI they used. If not, then you will have to try different amounts of virus particles but keep your cell number constant (i.e. 1:1, 2:1, 5:1, 10:1, 20:1, 40:1 - this is virus particles:cells). You can reduce your cell numbers way down so you don't have to use as much virus with the high MOIs.
Hi Tom Masi: Thanks a lot. But the total vial contains only 200,000 virus particle in 200 ul volume which cost around 700$. Does it feasible to use all virus (1:1) to transfect 200,000 cells? Besides do you think 200,000 cells are enough for getting protein after lysis for Western blot? Or is there any ways I can increase the number of cells after transfections to get sufficient number for Western Blot?
Hi Juan:
I have not used electroporation before. Does it require any special apparatus? I will glad if you share your protocol, then I can check if we have opportunity to do that in our lab or not.
Thanks in advance
Hi Shariful,
at least to test whether your virus with sc-108083 works for your cells, you can check GFP under fluorescence microscope or FACS, than you do not need more than 20.000 cells to transfect.
If then costs are an issue, you can of course produce lenti- or retrovirae in your lab. You can get the necessary vectors for low cost from addgene.com. Its laborious, though.
Hi Shariful,
I wouldn't recommend using all of your virus for a 1:1 MOI (200,000 cells). You most likely will not have good transduction efficiency. You can transduce a very small number of cells such as 1,000 with different MOIs (1:1, 2:1, 5:1, 10:1, 20:1, 40:1) and use less than half of your total amount of virus. After transduction you can just let them grow until you have sufficient numbers of cells to do your Western blot. The lentivirus will integrate into the host genome and should be stable for some time.
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