Reactive dyes for living cells: Applications, artefacts, and some comparisons with textile dyeing

An inclusive chemical definition of “reactive” dyeing of textiles is introduced, encompassing the CI Azoic, CI Mordant, CI Reactive, CI Sulphur and CI Vat dye application classes. Such reactive dyeing increases fibre retention of dye and makes application practically possible. The analogous application of dyes and fluorescent probes as microscopic stains in biology and medicine is outlined, focussing on using reactive fluorescent probes with living cells. Parallels with textile dyeing are noted, eg, enhanced probe retention and facilitation of probe application. However, the primary purpose of using reactive probes with live cells is detection of properties of biological systems: to identify biological structures and chemical/biochemical contents; assess biological functions and physicochemical properties; and determine changes in locations of cells and cell components. Problems occurring with such probes are outlined, particularly the problematic character of many standard protocols, and localisation artefacts arising with reactive probes whose reactant and product species are physiochemically significantly different. This latter problem is explored via a case study of possible reactant/product artefacts with probes for reactive oxygen species. Comparison of experimental observations of probe localisations with the localisations predicted using quantitative structure activity (QSAR) modelling indicates that such artefacts can occur with a significant proportion of chemically diverse, widely used, commercially available probes, as well as with experimental compounds reported in the literature. A graphical flowchart is provided to assess possible occurrence of reactant/product artefacts arising with reactive fluorescent probes localising in various organelles of living cells.