Hi everyone,
I am using Jurkat cells for transduction with the lentivirus generated with SBI packaging mix.
I used spinfection but did not work no gfp in gfp-control well.
Second time I have added the virus to cells-media-poybrene comples and incubated @37 temp. I could see hardly 7-10 cells expressing gfp.
my question is
1. does anybody tried transduction in Jurkat cells? how to get good infection efficiency with lentivirus?
2. How to get good viral titer?
Any suggestion?
I have experience in adhesion cell, but i can give you some suggestion:
1- TITER: seed 293t cells in 6 well plates and add dilution of virus suspension (100-50-25-10-1 ul of virus + one well with no virus as negative control). After 48h count GFP positive cells (cytoflorimetry): you should have a linear decrease of the cell number dependent on viruses added. this is your titer calculation
2-You need also calculate the MOI (multiple of infection), which consists in a ratio between your number of cells and the amount of viral particle. You need to calculate it empirically and for some experiment you need to keep in eye how many copy number you want add in each cell (for crispr the MOI is 1.5! ).
3-Polybrene is tossic for my cells. Have a try with that, but it will not make so much difference anyway.
Thank you Miguel Hermida, that is a good idea of keeping overnight incubation with virus. I hope you are keeping mix in 37 degree incubator right?
In my case I see polybrene (8 ug/ml) is toxic to cells.
Dear Hermuda,
I would like to confirm the following,
1. did your Jurkat cells were healthy (no stress) after overnight incubation with meida-virus-polybrene-cells complex?
2. how many cells and what is the volume of media have you used for Overnight incubation for transduction?
3.concentration of polybrene 8ug/ml- is this the same concentration you have used?
Thank you very much in advance...
Hi Mohammad,
What cell line are you using for virus production? 293T? When are you collecting the supernatant? I would suggest 2 collections post-transfection at 48 and 72 hour. You could try adding 6mM sodium butyrate 3 hours after transfection to increase titer. You could also try to concentrate your virus if you have access to an ultracentrifuge or you can use another method such as PEG-IT concentrator or other commercial reagents.
Regarding your Jurkat transduction, I would suggest doing spinfection at 2300RPM, 32C for 90 min. Following spinfection, pipet to mix and incubate for another 6 hours or overnight. You could add 5ug/ml polybrene for increased efficiency and very low affect on viability.
Hope this helps!
Ahmar
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