pkh-26 and fluorexon

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.

Topics: Animals; Benzimidazoles; Cell Division; Cell Membrane; Cell Movement; DNA; Fluoresceins; Fluorescent Dyes; Humans; Lymphocytes; Mice; Organic Chemicals; Succinimides

Gap junction intercellular communication (GJIC) is involved in several aspects of normal cell behaviour, and disturbances in this type of communication have been associated with many pathological conditions. Reliable and accurate methods for the determination of GJIC are therefore important in studies of cell biology. (Tomasetto, C., Neveu, M.J., Daley, J., Horan, P.K. and Sager, R. (1993) Journal of Cell Biology, 122, 157-167) reported some years ago the use of flow cytometer to determine transfer between cells of a mobile dye, calcein, as a measure of cell communication through gap junctions. In spite of this being a method with potential for quantitative and reliable determination of GJIC, it has been modestly used, possibly due to technical difficulties. In the present work we have illustrated several ways to use flow cytometric data to express cell communication through gap junctions. The recipient cells were pre-stained with the permanent lipophilic dye PKH26, and the donor cell population were loaded with the gap junction permeable dye, calcein. We show that the method may be used to measure the effect of chemicals on GJIC, and that the information is reliable, objective and reproducible due to the large number of cells studied. The data may give additional information to that obtained with other methods, since the effect observed will be on the establishment of cell communication as compared to what is observed for microinjection or scrape loading, where the effect is on already established communication. This is probably the reason for the more potent effects of DMSO on GJIC measured by the present method than on already existing GJIC measured by microinjection or quantitative scrape loading. We also show that the problem related to the mobile dye calcein not being fixable with aldehydes will not affect the results as long as the cells are kept on ice in the dark and analysed by flow cytometer within the first hours after formalin cell fixation.

Topics: Animals; Cell Communication; Cell Line; Chlordan; Epithelial Cells; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Gap Junctions; Humans; Insecticides; Kidney; Liver; Organic Chemicals; Rats