How to reduce cell autofluorescence? - (Jun/18/2007 )

Hi, I'm growing some mouse fibroblasts for fluorescence imaging and unfortunately the cells that I'm working with seem to exhibit a whole lot of auto fluorescence ever when excited @ 532 and 594nm. I was wondering if anyone has any idea as to how I could try to lower the auto fluorescence through culturing techniques. I'm currently using DMEM medium w/o phenol red, and FCS ( I image in serum-free medium.)
much thanks!

Hello Ender,

how long do you need your cells while imaging?

Maybe the time is short enough to keep them in PBS (with Calcium and Magnsesium) or Ringer Solution etc.?

Chakchel

A lot of people face autofluorescence issues with all sorts of techniques. This has prompted the development of longer-wavelength fluors that have emission wavelengths in the near-infrared range (680-800 nm). There is very little autofluorescence when you get up into these wavelengths, so the problems just dissappear without having to waste any time trying to figure out the cause and guess at solutions. I think more people will be migrating to this solution in time.

This may not be an option with your current equipment, but thought I'd mention it anyway. Something to consider when your lab purchases equipment in the future.

I can't think of any, but that doesn't necessarily mean they aren't out there.

The most red-shifted variants that I'm aware of emmit at about 640 nm. More info can be found on http://www.tsienlab.ucsd.edu/ (and more specifically http://www.tsienlab.ucsd.edu/Publications/...%20proteins.pdf and http://www.tsienlab.ucsd.edu/Publications/...eric%20red.pdf).