Q: Is there a positive control cell line that is easily infected by lentivirus?
A: We don’t have a control cell line, but we suggest using an easy to infect cell line such as 293T or our own 293LTV cell line. For ecotropic lentivirus we recommend NIH3T3 cells.
Q: How does your ViraDuctin™ Lentivirus Transduction Kit work?
A: ViraDuctin™ Lentivirus Transduction Kit is a reagent cocktail that uses technology developed at Harvard Medical School and can increase transduction efficiency 2-6 fold compared to polybrene. This product uses two chemical polymers that will form a complex with the virus and will settle onto the cells, increasing the local virus concentration at the cell surface.
Q: Is the ViraDuctin™ kit cell type specific?
A: Because the technology used in our ViraDuctin™ Lentivirus Transduction Kit does not rely on a cell receptor, it will work with any cell type, as long as it is not toxic to the cells.
Q: Are the reagents toxic to certain cell types?
A: We have not tested this with every cell line and do not know if it will be toxic or cause stress to specific cell lines, however but we have found it to be toxic to some primary cells. It has not been found to be toxic to most cancer cell lines.
Q: How do I convert LP/ml to IFU in order to calculate MOI?
A: The optimal MOI is dependent on target cell type and is best found from a literature search. The MOI is expressed as a ratio of the number of IFU or TU added to the cells (IFU/cell or TU/cell). For example, if your cell line requires an MOI of 200, this would require 200 IFU per 1 cell. LP/mL is a measure of physical titer and will need to be converted to IFU/mL to determine the MOI. The calculated LP/mL values can be converted to IFU/mL by dividing by 100.
Q: What final concentration of blasticidin and puromycin are necessary for making stable cell lines?
A: When creating stable cell lines, the optimal antibiotic concentration that is toxic to your cells will need to be determined by performing an antibiotic titration combined with cell density titration. The concentration range for puromycin and blasticidin is usually between 1-10 µg/mL.
Q: How can I improve my transduction efficiency?
A: Here are some suggestions for increasing lentiviral transduction efficiency:
1. We recommend measuring the titer of your virus and using the recommended MOI for the target cell line you are using. If you are unable to titer your virus, an option is to increase the amount of viral supernatant when infecting target cells.
2. Concentrate the virus using one of the following methods:
a. A concentrator unit with a 100,000 kDa molecular weight cut off membrane.
b. Our Concentration and Purification kits (VPK-090, http://www.cellbiolabs.com/lentivirus-concentration-purification-kits-column-based), which can concentrate the virus up to 500 fold.
c. PEG precipitation: For example, add 1/2 volume of ice-cold 30% (wt./vol.) PEG (6000 MW) dissolved in 0.5 M NaCl to the viral supernatant, and incubate overnight at 4 C with occasional mixing. Virus-PEG mixtures are then centrifuged at low speed (3000 ×g) for 15 min in a prechilled rotor at 4°C. Remove the supernatant by aspiration and resuspend the pellets in cell culture media or desired buffer.
3. Avoid using antibiotics in the media when packaging your virus and use low passage number cells for infection. If you are using difficult to infect cells you may first want to try using 293 cells, which are easily infected.
4. You can consider using our ViraDuctin™ Lentiviral Transduction Kit or polybrene as an additive to improve transduction efficiency.