"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: Potential application at microgravity conditions in space

Background: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing t o remove the unbound dye. There are instances, however, when use of stain s olutions is undesirable-e.g., at microgravity conditions in space, where th e possibility of accidental spill (many dyes are known carcinogens) introdu ces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat . Methods: The "liquidless" staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels tha t have been presoaked in the stain solutions. Fluorochromes that have affin ity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoech st 33342) or to protein (sulforhodamine 101) were used to saturate the gels . The gel strips were placed over the prefixed cells or tissue sections dep osited on microscope slides and relatively low (20 g/cm(2)) pressure was ap plied to ensure the contact. The cells were also stained by using commercia lly available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by laser scanning cyto metry (LSC). Results: Satisfactory cell and tissue staining, with minimal background, wa s achieved after 10-20 min contact between the cells and gels. Optimal conc entrations of the dyes in the solutions used to presoak the gels was found to be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G(1), S, and G(2)/M cell populations and 2:1 ratio of G(2)/M to G(1) peak fluorescence. Indivi dual gels can be saturated with more than a single dye-e.g., to obtain diff erential DNA and protein staining. Cell staining with DAPI or PI in the gel atin-based mounting media led to high fluorescence background while stainin g with DAPI in "aqueous" medium was satisfactory. Conclusions: Relatively fast staining of cells or tissue sections on micros cope slides can be achieved by nonconvective dye diffusion using hydrated g els permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions. Cytometry 44:355-360, 2001. (C) 2001 Wiley- Liss, Inc.