I want to isolate AAV particles from tranfected adherent cells. Would appreciate a best working protocol to isolate AAV particles using the kit based method or any minmium time consuming method.
Muhammad Jahangir Shafi
Transfection: Transfect adherent cells (e.g., HEK293T) with AAV rep/cap plasmids and a transfer vector. Incubate for 48-72 hours. Harvesting: Collect the supernatant and/or detach the cells. Centrifuge at 1,500-3,000 x g for 10 minutes, then filter the supernatant (0.45 µm filter). Concentration (Kit-Based): Use a commercial AAV isolation kit (e.g., Thermo Fisher or Qiagen) for easy purification and concentration of AAV particles (takes 4–6 hours). Optional Ultracentrifugation: For higher purity, perform ultracentrifugation using a sucrose or iodixanol gradient (2-3 hours). Titering: Quantify AAV concentration via qPCR or ELISA
To isolate AAV particles from transfected adherent cells, the following protocol is effective and time-efficient:
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