Fluorescent dyes for lymphocyte migration and proliferation studies

Fluorescent dyes are increasingly being exploited to track lymphocyte migration and proliferation. The present paper reviews the properties and performance of some 14 different fluorescent dyes that have been used during the last 20 years to monitor lymphocyte migration. Of the 14 dyes discussed, two stand out as being the most versatile in terms of long-term tracking of lymphocytes and their ability to quantify lymphocyte proliferation. They are the intracellular covalent coupling dye carboxyfluorescein diacetate succinimidyl ester (CFSE) and the membrane inserting dye PKH26. Both dyes have the advantage that they can be used to track cell division, both in vitro and in vivo, due to the progressive halving of the fluorescence intensity of the dyes in cells after each division. However, CFSE appears to have the edge over PKH26 based on homogeneity of lymphocyte staining and cost. Two other fluorescent dyes, although not suitable for lymphocyte proliferation studies, are valuable tracking dyes for short-term (up to 3 day) lymphocyte migration experiments, namely the DNA-binding dye Hoechst 33342 and the cytoplasmic dye calcein. In the future it is highly likely that additional fluorescent dyes, with different spectral properties to CFSE, will become available, as well as membrane inserting fluorescent dyes that more homogeneously label lymphocytes than PKH26.