How much does Polybrene cost?
Usually the range of polybrene conc. 4-8 ug/ml, depending on your cell types. For fibroblast i use 8 microgram/ml but while I use Neuron progenitor cells I don’t use Polyrene (it is toxic for NPC).
How do you transduce a virus to a cell?
c. Transduction
- Thaw the lentivirus on ice. Mix 8 µl Polybrene (1 mg/ml aliquot) with 957 µl culture.
- The next day, exchange Lentivirus/Polybrene mixture by fresh culture medium. Incubate cells at standard cell culture conditions.
- concentrations range from 0.1-10 μg/ml. Replace the culture medium 48-72 hours.
How do you dilute Polybrene?
To prepare 1mg/ml Polybrene stock, weigh 50mg of the Polybrene powder and dissolve in 50ml 0.9%Nacl solution of Milipore water. This will give you (1mg/ml). Filter sterilize and make aliquots into small tubes. Store at -20 degrees.
Is polybrene toxic to cells?
Polybrene (Hexadimethrine bromide) is a polycation that neutralizes charge interactions to increase binding between the pseudoviral envelope and the cellular membrane. Excessive exposure to Polybrene can be toxic to some cells.
What is polybrene used for?
Polybrene is a cationic polymer that can greatly enhance the efficiency of the retroviral or lentiviral infection to the mammalian cells. It acts to neutralize the charge repulsion between virions and the cell surface, thereby increasing infection efficiency.
Is polybrene stable?
Polybrene solution is stable for 2 months at +4°C.
Do you need polybrene for transduction?
It is definitely possible to transduce your cells without the presence of polybrene or any other polycation.
How much polybrene do I add?
Add 0.5 mL of a single viral dilution to each well (each well gets one dilution, so a 6-well plate will hold 5 dilutions plus one ‘no virus’ control well)….Procedure.
| Dilution | Volume of Lentivirus (μL) | Volume of DMEM complete + 10 µg/mL polybrene (µL) |
|---|---|---|
| 1:5 | 300 | 200 |
| 1:10 | 150 | 350 |
| 1:50 | 30 | 470 |
| 1:100 | 15 | 485 |
What is lentivirus used for?
Use as gene delivery vectors Lentivirus is primarily a research tool used to introduce a gene product into in vitro systems or animal models. Large-scale collaborative efforts are underway to use lentiviruses to block the expression of a specific gene using RNA interference technology in high-throughput formats.
How is Polybrene used as a transfection reagent?
Polybrene with a DMSO shock is also used to mediate DNA transfer into a variety of cell types, such as CHO, chicken embryo fibroblasts, NINH-3T3 and Myeloid cells. Recombinant retroviral stocks are prepared by adding 5mls of growth medium with 5% serum to a near confluent monolayer of transfected retroviral packaging cells in a 100mm plate.
What should be the concentration of Polybrene in a well?
Since all the media in these wells was made with DMEM complete + 10 µg/mL polybrene, the final concentration of polybrene in each well should be 10 µg/mL. *Pro-Tip* Transducing too many cells relative to the number of virus particles reduces the transduction efficiency, resulting in massive cell death upon antibiotic selection.
How is Polybrene used in a retroviral infection?
The efficiency of retroviral infection is enhanced significantly, 100 to 1,000 fold in some cells, by including polybrene during the infection. Polybrene with a DMSO shock is also used to mediate DNA transfer into a variety of cell types, such as CHO, chicken embryo fibroblasts, NINH-3T3 and Myeloid cells.
How long to infect a cell with polybrene?
Infect cells with 2mls of the viral supernatant (or a dilution of the virus stock into 2mls) in the presence of 5ug to 10ug of polybrene per ml (final concentration). Incubate cells for 3 to 6 hours at 37°C. Add 8mls of complete medium.