I am packaging MSCV virus with Phoenix Eco, but titers are low - about 10^5, inspite of good transfection efficiency. As a result I have quite low and inconsistent results of fetal liver cells infection. I read that Plat E cells are considered to be more productive and stable comparing to Phoenix. Does anyone have an experience with both cell lines? Is it worth to try Platinum cells? Thanks
Can I ask if you're using the conditioned media from virus production or if you have concentrated your retrovirus using an ultracentrifuge or similar? And how many cells did you transfect to make your virus - 10cm plate, T-75 etc.
I use unconcentrated supernatant, filtered and supplemented with polybrene. I transfect several 10 cm plates with CaPO4. I apply fresh virus on cells and perform spinoculation. For titration I apply 1:1, 1:20 and 1:200 dilutions of virus to 3T3 cells and count GFP-positive 3d later. I do not concentrate virus since it is ecotropic, as I have read they do not tolerate concentration with centrifugation.
Might also want to check (in the off chance) of any low grade myco contamination in your cultures...cells (especially 293T and their derivatives) will appear to be fine (growth and morphology), but viral titers become lower than normal, not to mention altering the target cells as well.
Of course, there is the assumption that the MSCV bakcbone is good, your cells have maintained their packaging potential, etc etc. In general, I do a GFP virus on the side to make sure that my virus packaging system is working fine (i.e. decent titers and good infectivity).
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line