Cells were seeded into six-well plates at a density of 300,000 cells per well and allowed to attach for at least 6 hours. Spent media were discarded and replaced with 2.5 ml of fresh media, 500 μl of lentivirus, and polybrene (Santa Cruz Biotechnology, no. SC-134220) at a final concentration of 8 μg/ml (except when infecting UMRC-2 cells, when polybrene was omitted). Plates were centrifuged at 4000g for 30 min at 25°C and then incubated for 14 to 16 hours at 37°C. The supernatant was then removed and replaced with fresh media for 12 to 24 hours before the addition of selection antibiotics. Lentivirally infected 786-O cells were selected in media containing blasticidin (10 μg/ml), G418 (600 μg/ml), puromycin (2 μg/ml), or zeocin (100 μg/ml) as appropriate for the lentiviral drug resistance cassette. Lentivirally infected UMRC-2 cells were selected in media containing blasticidin (10 μg/ml) or G418 (1.8 mg/ml) as appropriate for the lentiviral drug resistance cassette. Lentivirally infected 769-P and A498 cells were selected in media containing blasticidin (10 μg/ml).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.