brief description of the protocol
day 1, 293T seeded (low passage and not grown to confluency)
day 2, cells transfected with 3rd generation lenti vectors
day 3, cells clearly expressing GFP, media changed to viral harvest media (high FBS, no antibiotics)
day 5, all cells have died and have lysed.
has anyone experienced this as I have no idea what has happened here, viral toxicity perhaps?
Hi Ben,
I typically collected my supernatants on days 3/4/5 (so 24/48/72h post-transfection), replacing the media each time. On the last day the cells were typically only loosely attached or were sloughing off. This was likely a combination of high confluence by day 5 and toxicity. The viral tires we got were good so I wouldn't be too concerned. Only difference is when we switched to harvest media ours contained pen/strep.
Thanks edwards,
Do you ever observe cell lysis at high viral titres? When I observed my cells 72 hours post transfection, some cells remained adherant amd normal looking whereas all of the non adherant cells have been lysed.
Im repeating the tramsfection this week with fresh reagents and media and hopefully I have more success this time!
I may try to change the media every day as you detail to avoid extremely high viral titres.
What lentivirus are you making, VSV-G? What transfection agent are you using, PEI, CaPO4, Lipofectamine etc
Are there antibiotics present during the transfection?
At 72h post-transfection the cells could be looking sickly especially if they're overconfluent - what colour is the media? However they should not be all dead and they shouldn't lyse.
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