Trypan blue dye enters viable cells incubated with the pore-forming toxin HlyII of Bacillus cereus

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.     

Determination of growth and lysis kinetics in plant cell suspension cultures from the measurement of esterase release

The death of Medicago sativa L. cells cultivated in a batch culture was investigated by measuring both the appearance of intact dead cells determined on the basis of the trypan blue (TB) dye exclusion, and the release of the cytoplasmic esterase activity into the culture medium upon cell death. Taking into account the strong instability of this released esterase activity, the total dead cell and lysed cell densities have been estimated. A mechanism for cell death and lysis is proposed and the specific rates of cell growth, death and lysis estimated. The specific rate of appearance of TB dead cells was low and essentially constant (0.25 day(-1)) during the first 8 days of the batch culture, and then increased above 1.5 day(-1) after 2 weeks of cultivation. Whereas no lysis occurred during the first seven days, this phenomenon occurred during the second period and accounted for about 20% of the total cell death by the end of the process. Thus, the viability determined by the trypan blue exclusion method appeared to be invalid after 7 days of culture. When lysis of viable cells is taken into consideration, the specific growth rate was significantly increased and growth was shown to continue for a further 8 days. Increased sensitivity of the cells to shear stresses and consequent cell lysis could be the result of a 35% increase in the cell size Copyright 1999 John Wiley & Sons, Inc.     

TREATMENT OF RAT PROXIMAL AND DISTAL COLONIC CELLS WITH SODIUM ORTHOVANADATE ENHANCES THEIR ADHESION AND SURVIVAL IN PRIMARY CULTURE

We have studied the effect of sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, on primary cultures of colonocytes and stromal cells. Everted proximal and distal colonic tissue of adult rats were disintegrated by a collagenase/dispase solution for 60 min at 37°C to prepare viable gland fragments and isolated cells. Cell preparations were inoculated onto plastic substratum or cytodex-3 microcarriers in a defined maintenance medium or in 1% fetal calf serum media. Incorporation of sodium orthovanadate (≥50 μm) in these media constantly enhanced the survival (cell enumeration and trypan blue exclusionP<0.05) and the adhesion (up to four-fold by crystal violet staining,P<0.01) of colonocytes (characterized by cytokeratin-18, transforming growth factor-α or alkaline phosphatase expression) and stromal cells. Removal of sodium orthovanadate from culture media restored cellular death processes. Incorporation of 10 mmn-butyric acid did not promote cell adhesion and survival except for distal cells exposed to 2 mm sodium orthovanadate. Besides studies in the regulation of anoikis in primary culture, the model will help to assay the influences of dietary and growth factors on the biology of non-cancerous colonic cells.     

Dead cell counts during serum cultivation are underestimated by the fluorescent live/dead assay

The live/dead fluorescent assay provides a quick method for assessing the proportion of live and dead cells in cell culture systems or tissues and is widely used. Dead cells are detected by the fluorescence produced when propidium iodide (PI) binds to DNA; PI and similar molecules are excluded from live cells but can penetrate dead cells because of their loss of membrane integrity. Here we investigated the effect of serum in the culture medium on the reliability of the method. We assessed viability of chondrocytes with/without serum using both a live/dead assay kit and also trypan blue staining. We found that after 2 days of culture, the DNA-binding dye PI could no longer detect dead cells if serum was present but they were readily detected in serum-free medium or if an inhibitor to DNase I was added to the serum-containing medium. Dead cells could be detected by trypan blue staining in all cultures. Hence dead cells are no longer detected as the DNase I present in serum degrades their DNA. DNA-binding dyes may thus not give a reliable estimate of the number of dead cells in systems that have been cultured in the presence of serum for several days.     

Analysis of cytotoxicity of melittin on adherent culture of human endothelial cells reveals advantage of fluorescence microscopy over flow cytometry and haemocytometer assay

Melittin, from the honeybee venom, is a membrane active protein, whose cytotoxicity to human endothelial cells has not been described yet. In this work, we studied its time-dependent cytotoxicity on human umbilical vein endothelial cells (HUVECs). Since HUVECs grow in culture as adherent cells, suspension of cells is required before measuring cytotoxicity with a haemocytometer or flow cytometry. Therefore, we also tried to discover whether the result of cytotoxicity tests of melittin is influenced by the preparation of the cell suspension. For this purpose, we compared the results of haemocytometer-based trypan blue assay and flow cytometry using 7-aminoactinomycin D (7-AAD) with results of fluorescence microscopy using 7-AAD and 4', 6-diamidino-2-phenylindole (DAPI). Melittin over 60 min exposure evoked a rapid decline in the survival of HUVEC. After 60 min exposure to melittin, the phase contrast microscopy demonstrated massive necrosis in the remaining attached cells. Fluorescence microscopy detected both viable and non-viable cells in adequate proportions at all exposure times, whereas haemocytometer-based assay and flow cytometry highly underestimated the percentage of non-viable cells or even failed to detect any dead cells. Our data clearly indicate that the induction of large-scale damage to adherent endothelial cells by melittin results in a loss of the majority of necrotic cells during sample preparation for flow cytometry or a haemocytometer-based assay. In the case of adherent cell culture, therefore, fluorescence microscopy was shown to be a more appropriate method for quantitative analysis of cell death caused by a fast-acting cytolytic toxin such as melittin.     

A new methodology for plant cell viability assessment using intracellular esterase activity

 A plant cell suspension culture of Alfalfa (Medicago sativa L.) was grown in a bioreactor using a batch procedure. The cytoplasmic esterase activity (EC 3.1) was extracted from the cells and measured during cultivation using fluorescein diacetate as the fluorogenic substrate. This enzymatic activity was conclusively found to be correlated to cell viability assessed with the membrane integrity test using the trypan blue dye. This new viability determination method is convenient, simple and can be reproduced because: (1) the difficult step of counting the cells when using the trypan blue exclusion method is avoided and (2) the esterase activity level per viable cell constituted of numerous enzymes depends on cell viability but is independent of cellular metabolism. Received: 28 January 1999 / Accepted: 1 April 1999     

Simple detection method for distinguishing dead and living yeast colonies

A rapid and simple assay was developed for detection of yeast colonies containing dying or dead cells. Methylene blue, phloxin B, rose bengal and trypan blue at concentrations of 5-10 micromol l(-1) were shown to stain non-viable cells in colonies of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans and Filobasidium capsuligenum without staining or affecting the viability of living cells of the colonies.     

Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release

The death of the hybridoma VO 208 in a continuous culture at pH 7 and 6.8 was investigated by measuring both the appearance of visible dead cells which do not exclude the trypan blue dye and the release of lactate dehydrogenase (LDH) in the culture medium. The intracellular LDH was found to be completely released either when live cells lysed or when they were transformed into visible dead cells. No significant lysis of blue dead cells could be observed at the two different pH. Using a LDH balance over the culture system, cell lysis was found negligible at pH 7, but accounted for 20% of the total cell death at pH 6.8. A methodology is proposed to evaluate the rate constants of hybridoma lysis and total death. For the investigated cell line in continuous culture, the calculated total cell death rate constant was found to increase from 0.002 h^–1 to 0.01 h^–1 when decreasing the pH from 7 to 6.8.Abbreviations D dilution rate (h^–1) - k_b specific trypan-blue dead cells appearance rate (h^–1) - k_L specific lysis rate of viable cells (h^–1) - k_d specific death rate (h-1) - LDH₀ lactate dehydrogenase activity in the feed culture medium (IU.l^–1) - LDH lactate dehydrogenase activity in the outlet culture medium (IU.l^–1) - LDH_i intracellular lactate dehydrogenase activity of viable cells (IU.10^–9 cells) - r_LDH total rate of LDH release (IU.h^–1.L^–1) - r_b transformation rate of viable cells into blue dead cells (10⁹ cells.h^–1.L^–1) - x_v viable cell concentration (10⁹ cells.l^–1) - x_b trypan-blue dead cell concentration (10⁹ cells.l^–1)     

Viability measurements of hybridoma cells in suspension cultures

Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability. The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.     

Differentiation of genes extracted from non-viable versus viable micro-organisms in environmental samples using ethidium monoazide bromide

Differentiation of DNA derived from viable or non-viable microorganisms within mixed microbial communities continues to be one of the greatest challenges in molecular studies of environmental samples. A novel method developed for microbial food pathogens is tested here on environmental samples. This technique involves the use of ethidium monoazide bromide (EMA) for the distinction of live/dead cells. In non-viable cells EMA intercalates into the DNA which prevents amplification by PCR. We adapted and evaluated the EMA technique for soil, elemental sulfur and river biofilm samples. Quantitative PCR determined that EMA suppressed 99.99% of E. coli LKI gfp+ signal in non-viable cultures and 100.00% when the cultures were added to soil samples. The same technique was also successful at suppressing DNA amplification from spiked non-viable cells in elemental sulfur samples by 100.00%, but not in three Saskatchewan River biofilms. In sub Antarctic soil, EMA-Q-PCR was used to detect the prevalence of a functional gene, amoA, and this was closely correlated to nitrification activity measurements. The ability of EMA to differentiate between viable and non-viable populations in soil was confirmed by the similarity of the 16S rRNA denaturing-gradient-gel electrophoresis DNA fingerprint of EMA treated soil and the 16S rRNA cDNA fingerprint of non-EMA treated soil. The EMA technique effectively suppressed amplification of non-viable spiked controls, closely mirrored activity assays and yielded community composition profiles similar to rRNA techniques. The use of EMA in soil effectively suppressed amplification of non-viable organism DNA, however it was not effective in biofilm samples and EMA partially inhibited amplification of viable organism DNA in elemental sulfur samples.     

The distribution of cells killed by Trichinella spiralis in the mucosal epithelium of two strains of mice

Dead and dying cells were localized by light microscopy in the mucosal epithelium of the intestine of an outbred strain (CD1) and an inbred strain (B10A) of mice by vital staining with the dye, trypan blue. In whole mounts of the intestinal wall, trails, or variable-sized clusters of blue-stained cells were seen throughout the course of infection and in mice given a range of inoculum levels. In CD1 mice, irregular trails of dead cells were seen in the intestine floor and clusters of them along the villi. In B10A mice, dead cells were seen only as trails or clusters in the intestinal floor. The results suggest that worms move through the epithelium only in the intestinal floor. Cells killed by this activity may be sloughed from the epithelium more rapidly by B10A mice than by CD1 mice where the dead cells migrate up villi before being sloughed.     

Proliferation and death of cultured fetal neocortical neurons: effects of ethanol on the dynamics of cell growth

Neuronal number in the mature CNS is determined by the balance of cell proliferation and death. The effects of ethanol on cell proliferation and death were examined in primary cultures of neocortical neurons derived from 16-day-old rat fetuses. The cells were treated with ethanol (0 or 400 mg/dl) and examined for (1) immunohistochemical identity, (2) cell cycle kinetics using a cumulative bromodeoxyuridine labeling technique, (3) viable cell number via a trypan blue assay, and (4) the incidence of cell death with terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase 3 immunhistochemistry. After two days in culture, most (>85%) cells expressed a neuron-specific antigen(s) whether or not ethanol was added to the culture medium. Ethanol affected the proliferation of the cultured cells, e.g., the length of the cell cycle was greater in the ethanol-treated cells than in controls. The number of trypan blue-negative (viable) cells was profoundly decreased by ethanol exposure. This decrease was accompanied by increases in the frequencies of TUNEL- and caspase 3-positive cells and of cells exhibiting nuclear condensations. Thus, ethanol decreases the number of viable cells in vitro by slowing cell proliferation and increasing the incidence of cell death. The expression of the death indices in untreated cultures is most consistent with a single (apoptotic) pathway of cell death, rather than simultaneous apoptotic and necrotic modes of death. Furthermore, it appears that ethanol initiates an apoptotic death among cultured cortical neurons.     

定点整合人Bcl-2基因的CHO/dhfr-细胞株的建立

构建重组载体质粒pMCEfrt—Bcl-2,利用FIp—In^TM定点重组系统,在CHO—dhfr^-细胞内定点整合人Bcl-2基因,通过Western印迹检测重组细胞Bcl-2蛋白的表达。通过流式细胞仪和DNA Ladder检测在高NH4C1条件下细胞的凋亡情况;用台盼蓝染色检测在无血清IMDM培养基中细胞的活细胞数目和活细胞比例。结果获得了稳定表达Bcl-2基因的细胞株CHO—Bcl-2,该细胞株能高水平表达Bcl-2蛋白。在无血清培养过程中,CHO—Bcl-2细胞比对照细胞保持高约15％的活细胞比例,细胞总数高25％。CHO-Bcl-2在高NH4^＋（50mmol／L）培养条件下具有较低的凋亡水平。建立了能够高表达Bcl-2基因并具有一定的抗凋亡能力的重组CHO／dhfr^-细胞株。     

Influence of hormones and growth factors on viability,DNA, and protein content of adult hepatocytes in primary culture

Summary The survival of adult rat hepatocytes in monolayer culture was studied in the presence of different hormones (neurotensin, oxytocin, thyrotropin releasing hormone, luteinizing hormone releasing hormone, cholecalciferol, bradykinin, substance P, aldosterone, melanocyte stimulating hormone, 3,3′,5-triiodo-1-thyronine, corticosterone, human growth hormone, glucagon, insulin, progesterone, testosterone, estradiol, and dexamethasone phosphate) or growth factors (fetal bovine serum). For this purpose trypan blue exclusion, lactate dehydrogenase, and DNA and protein content were measured at 24 and 72 h of culture. 10⁻⁷ M Dexamethasone, a mixture of eight hormones, 10% fetal bovine serum, and a combination of the latter two supplements caused a more than 64% higher DNA content at 72 h when compared to control cultures. A striking agreement of these results with changes of lactate dehydrogenase leakage was observed, whereas trypan blue exclusion gave erratic results. Considerable changes of cell arrangement apparently specific for each supplement were ovserved by low magnification microscopy. It is concluded that glucocorticoids and fetal bovine serum have an outstanding effect on cell viability and that DNA or protein content or both are reliable indicators of cell viability in amitotic cultures.     

Long-term cell culture of two differentiated cell types from the liver of larval and adult Xenopus laevis

Summary Two distinct types of cells were derived from organ cultures of liver from adult and larval Xenopus laevis. Each type was isolated in clonal cell culture. Several media were compared with respect to support of epithelioid outgrowths from explants and support of growth of epithelioid colonies in cell culture. Ultracentrifuged embryo extract promotes the growth of all cell types, but the particulate fraction is also required for the maintenance of the epithelioid morphology of larval cells. In these media it was possible to maintain some epithelioid cell cultures for over 6 months. The identity and retention of some specialized functions of both cell types were demonstrated on larval cells. One cell type contained PAS-stainable, amylase-sensitive granules that increased in amount after treatment with glucocorticoids. This same type was shown by histochemical methods to contain phosphorylase, glucose-6-phosphatase, and dexamethasone-inducible tyrosine aminotransferase, and is considered to be a hepatocyte. The second type appears to be a sinusoidal cell, since it phagocytosed trypan blue and stained positively for acid phosphatase.     

A comparative study of cell death using electrical capacitance measurements and dielectrophoresis

The changes in the dielectric properties of cells that occur during their exposure to various lethal environmental stresses were measured using both dielectric spectroscopy and dielectrophoresis. It is shown that the dielectric properties of both dying and dead yeast cells were strongly dependent on the method used to induce cell death. Methods which directly affected the membrane permeability, and consequently the membrane conductivity and internal conductivity, resulted in large changes in the suspension capacitance and dielectrophoretic behaviour, whilst methods which affected the cell interior but had little effect on the cell membrane resulted in few or no changes in the dielectric properties of the cells. The findings indicate that, depending on the method by which cell death is induced, dielectric spectroscopy may not always be able to observe differences between viable and non-viable cells, and that dielectrophoresis will not always be able to separate viable from non-viable cells.     

Quantification of metabolically active biomass using Methylene Blue dye Reduction Test (MBRT): measurement of CFU in about 200 s

Quantification of viable cells is a critical step in almost all biological experiments. Despite its importance, the methods developed so far to differentiate between viable and non-viable cells suffer from major limitations such as being time intensive, inaccurate and expensive. Here, we present a method to quantify viable cells based on reduction of methylene blue dye in cell cultures. Although the methylene blue reduction method is well known to check the bacterial load in milk, its application in the quantification of viable cells has not been reported. We have developed and standardized this method by monitoring the dye reduction rate at each time point for growth of Escherichia coli. The standard growth curve was monitored using this technique. The Methylene Blue dye Reduction Test (MBRT) correlates very well with Colony Forming Units (CFU) up to a 800 live cells as established by plating. The test developed is simple, accurate and fast (200 s) as compared to available techniques. We demonstrate the utility of the developed assay to monitor CFU rapidly and accurately for E. coli, Bacillus subtilis and a mixed culture of E. coli and B. subtilis. This assay, thus, has a wide applicability to all types of aerobic organisms.     

Co-culture of primary pulmonary cells to model alveolar injury and translocation of proteins

Summary Primary rat alveolar type II cells and early passage rat lung fibroblasts were co-cultured on opposite sides of a collagen-coated polycarbonate filter. This is an approach to “model”, in part, an alveolar wall to study mechanisms of cytotoxicity and translocation of bioactive materials from the alveolar space to the lung interstitium. Type II cells were recovered from adult rat (Fischer 344) lungs by enzyme digestion and “panning”. Lung fibroblasts were separated from the same species, cultured initially in 10% fetal bovine serum and used in the co-culture system at early passage. The type II cells formed a monolayer of defferentiated epithelium which provided a barrier on the upper side of the collagen (human type IV)-coated filter. The fibroblasts on the bottom of the filter replicated logarithmically in the presence of serum, could be rendered quiescent in defined medium and then returned to rapid growth phase with the reintroduction of serum. The intact epithelial monolayer excluded trypan blue, albumin, platelet-derived growth factor, and alpha₂-macroglobulin from the lower compartment of the culture chamber. Altering the integrity of the monolayer by a variety of means allowed translocation of these materials through the collagen-coated filters. Particularly interesting was the effect of taurine chloramine which caused subtle changes in the alveolar epithelium and allowed subsequent translocation of albumin. In addition, we showed that rat alveolar macrophages remain viable with some spreading on the surface of the epithelial monolayer. This co-culture system will have future application in the study of how reactive oxygen species might affect the epithelial barrier, and whether macrophage-derived growth factors can influence fibroblast proliferation if the monolayer is intact or injured.     

Growth characteristics of human esophageal epithelial cells in primary explant and serial culture

Summary Growth characteristics of human esophageal epithelial cells have been determined in primary explant and serial culture. Normal human esophagus was obtained from donor patients in a heart/lung transplantation program; tissue obtained at autopsy (6 to 22 h after death) was not viable. When mucosal specimens (1.5 mm²) were explanted on a plastic surface and attached with a plasma clot, 35% of explants detached from the surface within 48 h. The addition of epsilon amino caproic acid (EACA) to the culture medium increased explant attachment of 93% (P<0.001). Outgrowth kinetics were similar in both the presence and absence of EACA. No advantage of human serum over nonhuman sera was observed in primary culture. Esophageal epithelium could be frozen in 10% dimethyl sulfoxide without affecting growth kinetics. Addition of dexamethasone (DEX) significantly altered esophageal cell morphology in primary culture and increased viability on serial culture. Studies of pH revealed an optimum at pH 7.4 with significantly decreased growth occuring at 6.8 and no growth at 6.2. Esophageal cells in primary explant cultures could be released by trypsin and passaged two additional times with an eightfould increase in total number. An increased rate of attachment and multiplication was observed for cells plated on a collagen substrate compared to platic. The addition of EACA and DEX to the culture media and the subculture on a collagen substrate provide a method for the isolation and serial cultivation of human esophageal cells from biopsy-sized specimens of normal esophageal epithelium. Supported in part by Grant AM—14121 of the United States Public Health Service. A preliminary report of this work appeared in Clin. Res. 30: 93A; 1982.     

Rapid Microscopy and Use of Vital Dyes: Potential to Determine Viability of Cryptococcus neoformans in the Clinical Laboratory

Background

Cryptococcus neoformans is the commonest cause of fungal meningitis, with a substantial mortality despite appropriate therapy. Quantitative culture of cryptococci in cerebrospinal fluid (CSF) during antifungal therapy is of prognostic value and has therapeutic implications, but is slow and not practicable in many resource-poor countries.

Methods

We piloted two rapid techniques for quantifying viable cryptococci using mixtures of live and heat-killed cryptococci cultured in vitro: (i) quantitative microscopy with exclusion staining using trypan blue dye, and (ii) flow cytometry, using the fluorescent dye 2′-7′-Bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM). Results were compared with standard quantitative cryptococcal cultures. Quantitative microscopy was also performed on cerebrospinal fluid (CSF) samples.

Results

Both microscopy and flow cytometry distinguished between viable and non-viable cryptococci. Cell counting (on log scale) by microscopy and by quantitative culture were significantly linearly associated (p<0.0001) and Bland-Altman analysis showed a high level of agreement. Proportions of viable cells (on logit scale), as detected by flow cytometry were significantly linearly associated with proportions detected by microscopy (p<0.0001) and Bland-Altman analysis showed a high level of agreement.

Conclusions

Direct microscopic examination of trypan blue-stained cryptococci and flow-cytometric assessment of BCECF-AM-stained cryptococci were in good agreement with quantitative cultures. These are promising strategies for rapid determination of the viability of cryptococci, and should be investigated in clinical practice.