Trypan Blue Dye Enters Viable Cells Incubated with the Pore-Forming Toxin HlyII of Bacillus cereus

Abstract

Trypan blue is a dye that has been widely used for selective staining of dead tissues or cells. Here, we show that the pore-forming toxin HlyII of Bacillus cereus allows trypan blue staining of macrophage cells, despite the cells remaining viable and metabolically active. These findings suggest that the dye enters viable cells through the pores. To our knowledge, this is the first demonstration that trypan blue may enter viable cells. Consequently, the use of trypan blue staining as a marker of vital status should be interpreted with caution. The blue coloration does not necessarily indicate cell lysis, but may rather indicate pore formation in the cell membranes and more generally increased membrane permeability.

Rhamnose‐coated superparamagnetic iron‐oxide nanoparticles: an evaluation of their in vitro cytotoxicity, genotoxicity and carcinogenicity

“…This exception may be due to the higher ability of Trypan Blue to enter the U251 cells which have more cell membrane damages (Fig. 4A) (Tran et al, 2011).…”

Functional Analysis of the Exocyst Subunit Sec15 in Candida albicans

“…Plates were incubated at 30°C for 24 h, and colonies were counted and graphed in logarithmic scale. To further assess cell viability, the trypan blue exclusion assay was performed as described previously (18). Trypan blue is a negatively charged diazo dye that cannot pass through the cell membrane unless the membrane is damaged.…”

“…Trypan blue is a negatively charged diazo dye that cannot pass through the cell membrane unless the membrane is damaged. Therefore, all the cells that exclude the dye are considered viable, and dead cells (with damaged membranes) are stained blue and can be easily observed under the microscope (18). Briefly, a subculture from overnight cells was grown in YPD medium at 30°C for 5 h with or without DOX.…”

“…To further confirm that the tetR-SEC15 strain remained viable for up to 5 h of incubation under the restrictive conditions (with DOX), we analyzed cell viability using a trypan blue exclusion assay. Trypan blue passively diffuses through the permeabilized cell membrane, a result of increased membrane permeability that accompanies cell death, but remains excluded from cells with an intact cell membrane (18). Thus, inviable cells stain blue when incubated with trypan blue whereas then for 2 h with or without DOX in the presence of chitinase.…”

Pathogenic Potential of Bacillus cereus Strains as Revealed by Phenotypic Analysis

“…Cytotoxicity of culture supernatants was evaluated by the trypan blue dye method (17,18). The pathogenic strains (FP and C) were highly toxic to human cells (more than 80% of the strains), whereas cytotoxicity was detected for only 12% of the NP strains (P Ͻ 0.001; Fig.…”