Q: What is the difference between the three Platinum cell lines?
A: The Platinum cell lines are used to package retrovirus and they differ in the envelope protein that is expressed. Here is information about each of the Platinum cell lines:
1. Platinum-A cells express MMLV-Gag-Pol and an amphotropic envelope protein and require transfection of only an expression vector containing your gene of interest to produce retrovirus. Amphotropic virus can infect mammalian cells.
2. Plat-E cells express MMLV-Gag-Pol and an ecotropic envelope protein. These cells require transfection with just an expression vector and can only infect mouse and rat cells.
3. The Plat-GP cells stably express MMLV Gag-Pol and can be co-transfected with pCMV-VSVG and an expression vector to generate VSVG pseudotyped retrovirus, which can infect any cell type.
Unlike VSVG pseudotyped virus, amphotropic and ecotropic viruses are not very stable, and can't handle ultracentrifugation. Please see the selection guide on our website for more information on the target cell species that can be infected by retrovirus.
Q: What is the typical titer of retrovirus packaged with Platinum cells?
A typical titer when using a Platinum packaging cell line is 10^7 TU/ml.
Q: Should I use Plat-E cells to make retrovirus for infection of mouse cells?
A: If you will only be infecting mouse cells, you may consider packaging ecotropic virus, which will only infect mouse and rat cells. If you plan on infecting other cell types, you can consider packaging amphotropic virus, which will infect mammalian cells, or pantropic virus, which will infect most species. Ecotropic virus is safer to work with because it will not infect human cells, but it is less stable than pantropic virus and cannot handle ultracentrifugation procedures used for concentration and purification.
Q: Will Platinum cells give a better yield than 293T cells?
A: The Platinum cell lines will give a higher titer and higher transfection efficiency than 293T or 293RTV cells and will be easier to transfect because Plat-E requires just one plasmid. Despite this, 293T and 293RTV cells can still produce a good titer. A typical titer for retrovirus under optimized conditions is 10^6 TU/mL with 293T cells and 10^7 TU/mL with Plat-E cells.
Q: Can the Platinum cells be cultured in DMEM containing high glucose?
A: It is fine to use any DMEM, including high glucose DMEM, for culturing Platinum cells. We use DMEM that contains high glucose (4.5 g/L) and L-glutamine.
Q: Should Platinum cell lines be cultured on coated plates to increase cell adherence?
A: Platinum cells can be cultured on standard tissue culture treated plates, and it is not necessary to use coated plates.
Q: How should I maintain Platinum cell cultures?
A: Platinum cultures should always be split when cells become confluent and should never be split more than 1:6.
Q: Why did these cells not adhere after thawing?
A: It is normal with the Plat-E cells to initially look very unhealthy and to take awhile to adhere after thawing, unlike most other cell lines that recover quickly. Detachment is normal during the first three days of culture, but if left alone during this time the cells will recover and attachment will improve. The culture should be continued until attachment improves. Once the culture has recovered and the cells have reached confluency, split the culture 1:4 and the cells should be healthy afterwards and look like 293T cells.
Q: These cells detach from the plate very easily.
A: The Platinum-A cells (RV-102) are derived from 293T cells and share similar morphology and growth characteristics. Both cell lines are easily detached and care should be taken not to shake or hit the flask.
Q: Are the Platinum cells compatible with my expression vector?
A: The Platinum cell lines are compatible with any MMLV based expression vector including pBABE and pMX.
Q: How much total DNA should I use for transfection?
A: The total amount of DNA used for transfection will be determined by the transfection protocol that you are using. We recommend following the manufacturer’s protocol for the reagent you are using, which should specify the total DNA amount based on the plate size.
Q: Should antibiotics be present in the media during the transfection?
A: Any antibiotics that are used to maintain stable cell lines should be avoided when packaging the virus because they may be toxic to target cells if carried over in the media during transduction.
Q: Can I use one of your Platinum Retroviral Packaging Cell Lines with a lentiviral expression vector containing my gene?
A: Even though lentivirus is a sub-class of retrovirus, the structural genes of HIV and MMLV are different enough that they are not interchangeable. Therefore combining a lentiviral expression vector with a retroviral packaging cell will not produce a viable viral particle.