The University of Iowa Carver College of Medicine

Flow Cytometry Facility

GFP and Fixation

The following suggestions are from Derek Davies, Imperial Cancer Research Fund, on GFP and fixation.

Suggestions for Fixation

  1. Use a low concentration of paraformaldehyde (between 0.25% and 1% for as little as 15 minutes.
  2. There is no need to wash before running the samples.
  3. The fluorescence is maintained after mild denaturation or with aldehyde fixation but fully denatured GFP is not fluorescent, presumably because the chromophore part of the molecule is obscurred. So, one does need to avoid other things that may denature the molecule (e.g. extremes of temperature and pH).
  4. Pre-fixation in paraformaldehyde is also required if one also wants to assess cell cycle status with PI. (see Simultaneous Staining with GFP and Propidium Iodide for DNA Content for staining protocol)