Chemists focus on probes, biologists on cells—but who talks about probe-cell interactions? A critical account of the suboptimal reporting of novel fluorescent imaging probes, using lipid droplet stains as a case study

Many current reports in the scientific literature describe novel fluorescent probes intended to provide information on various structures or properties of live cells by using microscopic imaging. Unfortunately, many such reports fail to provide key information regarding the staining process. It is often the case that neither the necessary minimum technical detail (probe concentration, solvent and cosolute, temperature and time of staining, and details of post-staining washes) nor a discussion of the proposed staining mechanism are provided. Such omissions make it unnecessarily difficult for biomedical end-users to try out reported novel probes in their own laboratories. The validity of these criticisms is explored and demonstrated by a detailed analysis of 75 non-cherry-picked articles describing novel fluorescent probes for the detection of lipid droplets in live cells. This dataset also suggests that papers from journals with high journal impact factors or from better-known research groups are no more likely to provide better protocol information or discussion of the mechanism than papers from less prestigious sources. Comments on possible reasons for this suboptimal reporting are offered. The use of a suitable information/feature checklist, following best practice in many leading chemical and biological journals, is suggested as a mechanism for ameliorating this situation, with a draft checklist being provided

Curious about stains? Need help with staining? Asking for a friend? Try the Biological Stain Commission's online Stain Glossary

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Tetrazolium salts and formazan products in cell biology: viability assessment, fluorescence imaging, and labeling perspectives

For many years various tetrazolium salts and their formazan products have been employed in histochemistry and for assessing cell viability. For the latter application, the most widely used are 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and 5-cyano-2,3-di-(p-tolyl)-tetrazolium chloride (CTC) for viability assays of eukaryotic cells and bacteria, respectively. In these cases, the nicotinamide-adenine-dinucleotide (NAD(P)H) coenzyme and dehydrogenases from metabolically active cells reduce tetrazolium salts to strongly colored and lipophilic formazan products, which are then quantified by absorbance (MTT) or fluorescence (CTC). More recently, certain sulfonated tetrazolium, which give rise to water-soluble formazans, have also proved useful for cytotoxicity assays. We describe several aspects of the application of tetrazolium salts and formazans in biomedical cell biology research, mainly regarding formazan-based colorimetric assays, cellular reduction of MTT, and localization and fluorescence of the MTT formazan in lipidic cell structures. In addition, some pharmacological and labeling perspectives of these compounds are also described

Is Mercury Orange a selective stain for thiols?

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42852/1/10735_2005_Article_BF01005240.pd

Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate

Natural and synthetic phenazines are widely used in biomedical sciences. In dehydrogenase histochemistry, phenazine methosulfate (PMS) is applied as a redox reagent for coupling reduced coenzymes to the reduction of tetrazolium salts into colored formazans. PMS is also currently used for cytotoxicity and viability assays of cell cultures using sulfonated tetrazoliums. Under UV (340 nm) excitation, aqueous solutions of the cationic PMS show green fluorescence (λem: 526 nm), whereas the reduced hydrophobic derivative (methyl-phenazine, MPH) shows blue fluorescence (λem: 465 nm). Under UV (365 nm) excitation, cultured cells (LM2, IGROV-1, BGC-1, and 3T3-L1 adipocytes) treated with PMS (5 μg/mL, 30 min) showed cytoplasmic granules with bright blue fluorescence, which correspond to lipid droplets labeled by the lipophilic methyl-phenazine. After formaldehyde fixation blue-fluorescing droplets could be stained with oil red O. Interestingly, PMS-treated 3T3-L1 adipocytes observed under UV excitation 24 h after labeling showed large lipid droplets with a weak green emission within a diffuse pale blue-fluorescing cytoplasm, whereas a strong green emission was observed in small lipid droplets. This fluorescence change from blue to green indicates that reoxidation of methyl-phenazine to PMS can occur. Regarding cell uptake and labeling mechanisms, QSAR models predict that the hydrophilic PMS is not significantly membrane-permeant, so most PMS reduction is expected to be extracellular and associated with a plasma membrane NAD(P)H reductase. Once formed, the lipophilic and blue-fluorescing methyl-phenazine enters live cells and mainly accumulates in lipid droplets. Overall, the results reported here indicate that PMS is an excellent fluorescent probe to investigate labeling and redox dynamics of lipid droplets in cultured cells.Fil: Stockert, Juan C.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; ArgentinaFil: Carou, María Clara. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; ArgentinaFil: Casas, Adriana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaFil: Garcia Vior, María Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; ArgentinaFil: Ezquerra Riega, Sergio Dario. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Blanco, María M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; ArgentinaFil: Espada, Jesús. Universidad Bernardo O'Higgins; ChileFil: Blázquez Castro, Alfonso. Universidad Autónoma de Madrid. Facultad de Ciencias. Departamento de Biología; EspañaFil: Horobin, Richard W.. University of Glasgow; Reino UnidoFil: Lombardo, Daniel Marcelo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentin

Dyes & fluorochromes as imaging agents in biology and medicine - new opportunities for dyestuff and textile chemists

Dyes and fluorochromes are used in biology and medicine to generate informative microscopic and macroscopic images. This means that the presence, location, biological structure, chemical content and physiological function of cells, tissues and whole creatures can be investigated. A wide range of dyes, both textile and functional, are used in this way. It is suggested that the various groups of stakeholders involved in this approach-end-users in biomedicine; synthesizers, manufacturers & vendors of dyes; and regulators and standardizers-have much to gain by becoming significantly better connected with each other. Broadly speaking, textile dyers use colorants to make socks and shirts and ties more visually attractive, which is not an objective of professional concern to biologists or clinicians. Biomedical applications of colorants are commonly aimed at obtaining information about living systems, using imaging technologies. There are also non-imaging dye applications in which they are used as drugs or analytical reagents, but these are not considered here. Note: below, “dyes” often implies fluorochromes, whilst “biomedicine” is short for biology and medicine

Theory of histological staining and its practical implications

No abstract available

Editorial note: celebration of the life and work of Dietrich Wittekind

No abstract available

How do histological stains work?

No abstract available