Slow virus vectors can infect both dividing and non dividing cells. It has many advantages such as a large capacity of transferred gene fragments, longer expression time of target genes, and less susceptibility to host immune responses; Therefore, lentiviruses have become one of the commonly used vector forms for expressing exogenous genes or exogenous shRNAs, and their applications are becoming increasingly widespread.
Introduction to lentivirus concentrate
EZ lentivirus concentrate is a product that rapidly concentrates lentiviruses. The concentrated virus solution can be obtained by incubating the uncondensed virus supernatant with this product at low temperature and centrifuging at low speed. Unlike traditional time-consuming and labor-intensive ultrafiltration and ultracentrifugation methods, this product is easy to operate, fast, safe, and has a high recovery rate.
Advantages of concentrated lentivirus solution
✔ High concentration ratio: The concentration volume can reach up to 240 times, for example, 120mL of virus supernatant can be concentrated to 500uL;
✔ High recovery rate: After testing, the virus recovery rate is as high as 92%;
✔ High concentration titer: After testing, the virus titer can often reach 108 or 109 after concentration with this product;
✔ High viral activity: The concentrated virus has better transduction effect.
Operation steps for concentrated lentivirus solution
① Collect the lentiviral supernatant from 293T cells at 48 and 72 hours, at 4 ℃, Centrifuge at 2000 g for 10 minutes.
② The supernatant was filtered through a 0.45 μ m membrane to remove cell debris. [Note]: The filter membrane should use cellulose acetate or polyethersulfone (PES) membrane with low protein adsorption. Never use nitrocellulose membrane (NC).
③ Mix according to the ratio of lentiviral filtrate volume: concentrated reagent volume=4:1, shake well, let stand overnight at 4 ℃, or incubate on ice for at least 3 hours. Prolonging the incubation time appropriately can improve the recovery rate of lentivirus.
④ After incubation, centrifuge at 4 ℃ and 3500 g for 15 minutes, carefully aspirate the supernatant.
⑤ Gently and repeatedly blow and aspirate the virus precipitate with an appropriate volume of PBS, resuspend the lentivirus.
⑥ Leave a small amount of virus solution to determine the titer, and store the rest after packaging at -80 ℃. [Note]: Due to the significant decrease in lentiviral activity caused by freeze-thaw cycles, please be sure to pack and store separately.
Precautions for concentrated lentivirus solution
This product is only for scientific experimental research and should not be used in clinical diagnosis, treatment, or other fields.
Please complete the experimental operations related to lentivirus in the biosafety cabinet.
Pay attention to thoroughly mixing the concentrated reagent and virus solution.
The growth status of cells is crucial for virus packaging, therefore it is necessary to ensure good cell growth status and fewer cell passages.
Do not repeatedly freeze and thaw the virus. If freezing and thawing are necessary, try not to exceed 3 times. About 10% of the virus is lost after each freezing and thawing. It should be packaged according to the amount of virus used each time and stored at -80 ℃. Try not to store for more than 6 months.
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