Dear all,
Recently I’ve been having a lot of issues with super high autofluorescence during live cell imaging (of what seems to be mitochondria). I have added a photo of what I mean. the problems are in the 488 and 561 channels (image was taken on a spinning disc confocal of live cells). The 640 channel does not have this problem…
I used to follow the exact same protocol and did not encounter this problem before. I have already tried the following:
· use new medium + new FCS and P/S
· thaw fresh cells (tried it in HEK293T and Hela-R19)
· use different well chambers I was wondering whether anybody has encountered this, and if so what a possible solution could be.
Thanks in advance, Jelle
Hi Jelle Schipper . Is your medium phenol red -free? Phenol red can quench the signal of some fluorescent dyes when used during live cell imaging. In addition, it is autofluorescent and can influence signal-to-noise ratio, especially if your fluorescent signal is weak.
Hi Jelle :),
I've imaged quite a lot HEK293T cells and have never seen such strong autofluorescence, especially just from one organelle...Can you tell us a little bit more about what you have done to the cells, your imaging medium, staining or purpose of your recordings, protocol...? Good luck!
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