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How to improve the lentivirus transduction efficiency in jurkat or...
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Kipha Valvoda
How to improve the lentivirus transduction efficiency in jurkat or...
You can increase efficiency of transduction using: 1) Polybrene (a cationic polymer that can greatly enhance the efficiency of the retroviral or lentiviral infection to the mammalian cells), 2) spinfection (centrifugation of the cells together with virus - increases contact between tumor cells and the virus) or 3) consecutive transduction (doing it twice on the same group of cells). Polybrene gives the best results, but does not work great with all cell types.
However, if the cells after transduction should be red, and you have 80-90% of them expressing mCherry, then the efficiency of transduction is great. I only got better efficiency when I was transducing HEK293 cells, and they are transduction control and very, very easy to transduce.
You can increase efficiency of transduction using: 1) Polybrene (a cationic polymer that can greatly enhance the efficiency of the retroviral or lentiviral infection to the mammalian cells), 2) spinfection (centrifugation of the cells together with virus - increases contact between tumor cells and the virus) or 3) consecutive transduction (doing it twice on the same group of cells). Polybrene gives the best results, but does not work great with all cell types.
However, if the cells after transduction should be red, and you have 80-90% of them expressing mCherry, then the efficiency of transduction is great. I only got better efficiency when I was transducing HEK293 cells, and they are transduction control and very, very easy to transduce.
I would recommend review/optimize your thawing step and conditions, there is a possibility you compromise the integrity of your lentiviral vector, also make sure your cells show a great viability (at least 80%). Jurkat cells are T cells and should be suspending not adherent making transduction a bit more tricky than adherent ones like HEK293. If you are planning to make cells stably expressing whatever your gene, you can use polybrene as Patrycja mentioned (usually at 8 ug/ml of your culture medium)
I would recommend review/optimize your thawing step and conditions, there is a possibility you compromise the integrity of your lentiviral vector, also make sure your cells show a great viability (at least 80%). Jurkat cells are T cells and should be suspending not adherent making transduction a bit more tricky than adherent ones like HEK293. If you are planning to make cells stably expressing whatever your gene, you can use polybrene as Patrycja mentioned (usually at 8 ug/ml of your culture medium)
Freeze thaw of viruses looses their functionality 30 to 50%. So try to use fresh lentivirus supernatant. If not, when you get virus out from -80C keep it on ice and let that to thaw by itself and should not allow virus to thaw in fast mode. Then slowly bring it to RT and use for transduction. Generally Kirk at and primary T cells will tranduce easily first give >80% and second >60% transduction efficiency. because I experienced the same. All the best.
Freeze thaw of viruses looses their functionality 30 to 50%. So try to use fresh lentivirus supernatant. If not, when you get virus out from -80C keep it on ice and let that to thaw by itself and should not allow virus to thaw in fast mode. Then slowly bring it to RT and use for transduction. Generally Kirk at and primary T cells will tranduce easily first give >80% and second >60% transduction efficiency. because I experienced the same. All the best.
You can increase efficiency of transduction using: 1) Polybrene (a cationic polymer that can greatly enhance the efficiency of the retroviral or lentiviral infection to the mammalian cells), 2) spinfection (centrifugation of the cells together with virus - increases contact between tumor cells and the virus) or 3) consecutive transduction (doing it twice on the same group of cells). Polybrene gives the best results, but does not work great with all cell types.
However, if the cells after transduction should be red, and you have 80-90% of them expressing mCherry, then the efficiency of transduction is great. I only got better efficiency when I was transducing HEK293 cells, and they are transduction control and very, very easy to transduce.
You can increase efficiency of transduction using: 1) Polybrene (a cationic polymer that can greatly enhance the efficiency of the retroviral or lentiviral infection to the mammalian cells), 2) spinfection (centrifugation of the cells together with virus - increases contact between tumor cells and the virus) or 3) consecutive transduction (doing it twice on the same group of cells). Polybrene gives the best results, but does not work great with all cell types.
However, if the cells after transduction should be red, and you have 80-90% of them expressing mCherry, then the efficiency of transduction is great. I only got better efficiency when I was transducing HEK293 cells, and they are transduction control and very, very easy to transduce.
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I would recommend review/optimize your thawing step and conditions, there is a possibility you compromise the integrity of your lentiviral vector, also make sure your cells show a great viability (at least 80%). Jurkat cells are T cells and should be suspending not adherent making transduction a bit more tricky than adherent ones like HEK293. If you are planning to make cells stably expressing whatever your gene, you can use polybrene as Patrycja mentioned (usually at 8 ug/ml of your culture medium)
I would recommend review/optimize your thawing step and conditions, there is a possibility you compromise the integrity of your lentiviral vector, also make sure your cells show a great viability (at least 80%). Jurkat cells are T cells and should be suspending not adherent making transduction a bit more tricky than adherent ones like HEK293. If you are planning to make cells stably expressing whatever your gene, you can use polybrene as Patrycja mentioned (usually at 8 ug/ml of your culture medium)
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I use VSV G-based lentiviral vector (PSPAx2,PMD2G and PLOX)
I use VSV G-based lentiviral vector (PSPAx2,PMD2G and PLOX)
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Freeze thaw of viruses looses their functionality 30 to 50%. So try to use fresh lentivirus supernatant. If not, when you get virus out from -80C keep it on ice and let that to thaw by itself and should not allow virus to thaw in fast mode. Then slowly bring it to RT and use for transduction. Generally Kirk at and primary T cells will tranduce easily first give >80% and second >60% transduction efficiency. because I experienced the same.
All the best.
Freeze thaw of viruses looses their functionality 30 to 50%. So try to use fresh lentivirus supernatant. If not, when you get virus out from -80C keep it on ice and let that to thaw by itself and should not allow virus to thaw in fast mode. Then slowly bring it to RT and use for transduction. Generally Kirk at and primary T cells will tranduce easily first give >80% and second >60% transduction efficiency. because I experienced the same.
All the best.
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