Trypan Blue - Adapting a Dye Used for Labelling Dead Cells to Visualize Pinocytosis in Viable Cells.

Abstract

BACKGROUND/AIMS: Trypan blue is routinely used in cell culture experiments to distinguish between dead cells, which are labelled by trypan blue, and viable cells, which are apparently free of any staining. The assumption that trypan blue labelling is restricted to dead cells derives from the observation that rupture of the plasma membrane correlates with intense trypan blue staining. However, decades ago, trypan blue has been used to trace fluid uptake by viable macrophage-like cells in animals. These studies contributed to the concept of the reticuloendothelial system in vertebrates. Trypan blue itself does not show a fluorescence signal, but trypan blue-labelled proteins do. Therefore, intracellular localization of trypan blue-labelled proteins could give a clue to the entrance pathway of the dye in viable cells. METHODS: We used fluorescence microscopy to visualize trypan blue positive structures and to evaluate whether the bactericide, silver, enhances cellular trypan blue uptake in the brain macrophage-like cell line, BV-2. The pattern of chromatin condensation, visualized by DAPI staining, was used to identify the cell death pathway. RESULTS: We observed that silver nitrate at elevated concentrations (≥ 10 µM) induced in most cells a necrotic cell death pathway. Necrotic cells, identified by pycnotic nuclei, showed an intense and homogenous trypan blue staining. Apoptotic cells, characterized by crescent-like nuclear chromatin condensations, were not labelled by trypan blue. At lower silver nitrate concentrations, most cells were viable, but they showed trypan blue labelling. Viable cells showed a cell-type specific distribution of heterochromatin and revealed a perinuclear accumulation of bright trypan blue-labelled vesicles and, occasionally, a faint homogenous trypan blue labelling of the cytoplasm and nucleus. Amiloride, which prevents macropinocytosis by blocking the Na+ / H+ exchange, suppressed perinuclear accumulation of dye-labelled vesicles. Swelling of cells in a hypotonic solution induced an intense intracellular accumulation of trypan blue. Cells exposed to a hypotonic solution in the presence of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), which blocks volume-regulated ion channels, prevented labelling of the cytoplasm and nucleus but did not affect labelling of perinuclear vesicles. CONCLUSION: In viable cells trypan blue-labelled vesicles indicate trypan blue uptake by macropinocytosis and trypan blue-labelled cytosol could indicate a further entry pathway for the dye, like activated volume-regulated channels. Accordingly, fluorescence microscopic analysis of trypan blue-labelled cells allows not only a discrimination between necrotic and apoptotic cell death pathway but also a discrimination between the mode of trypan blue uptake in viable cells - via pinocytosis or via activated volume-regulated ion channels - in the same preparation at the single cell level.

Bioceramic and polycationic biopolymer nanocomposite scaffolds for improved wound self-healing and anti-inflammatory properties: an in vitro study

“…60 Trypan blue negative cells made up about 96% of the cell population under control conditions, whereas trypan blue positive cells, which might be distinguished from necrotic cells by their pycnotic nuclei, made up about 4% of the cell population. 61 In contrast to mid concentrations (50 and 75 μL), the live/dead staining at 24 h revealed robust cell viability at lower and higher concentrations (25 and 100 μL). Compared to the control, all concentrations showed less viability.…”

Living Sample Viability Measurement Methods from Traditional Assays to Nanomotion

“…Dye exclusion assays determine the viability of suspension samples, with nonviable samples appearing in blue cytoplasm and living samples appearing in clear cytoplasm. Trypan blue [ 9 , 10 , 11 , 12 , 13 , 14 ] is a toxic assay for mammalian cells, and eosin [ 13 , 15 , 16 , 17 ], congo red [ 18 ], and erythrosine B stain assays [ 19 , 20 ] are nontoxic assays for mammalian cells. The principle of fluorometric assays is based on cellular esterases’ cleavage of a nonfluorescent compound into a fluorescent compound.…”

A review of the current state of natural biomaterials in wound healing applications

“…Based on this method, cells are mixed with trypan blue solution and placed on neobar slide or hemocytometer for cell counting and determination of concentration. Dead cells are seen in blue color due to damage to their membrane, which differentiates them from living cells ( Crowley et al, 2016 ; Kerschbaum et al, 2021 ). Over time, newer assays for cell viability were noticed.…”