Measuring plasma membrane fluidity using confocal microscopy

Membrane fluidity is a crucial parameter for cellular physiology. Recent evidence suggests that fluidity varies between cell types and states and in diseases. As membrane fluidity has gradually become an important consideration in cell biology and biomedicine, it is essential to have reliable and quantitative ways to measure it in cells. In the past decade, there has been substantial progress both in chemical probes and in imaging tools to make membrane fluidity measurements easier and more reliable. We have recently established a robust pipeline, using confocal imaging and new environment-sensitive probes, that has been successfully used for several studies. Here we present our detailed protocol for membrane fluidity measurement, from labeling to imaging and image analysis. The protocol takes ~4 h and requires basic expertise in cell culture, wet lab and microscopy.