Direct vs. indirect effects of trypan blue on the ectodermal cells of the 11.5-day rat embryo?

The effect of trypan blue on the 11.5-day rat conceptus after intravitelline vessel administration is described. For comparison, conceptuses injected with varying volumes of Hanks' BSS have also been studied. Trypan blue significantly retarded the growth and development of conceptuses after 6 hours incubation in vitro. The SEM revealed rounded ectodermal cells, some of which appeared disrupted. These cells seemed to cause some of the intersomitic grooves to disappear, making a number of the somites indistinct from the outside. Unlike cells of uninjected embryos, the surfaces of the affected ectodermal cells lacked microvilli and their perimeters were lined with microvilli-like structures which appeared matted together. It was concluded that trypan blue affected the embryo directly probably by disturbing its fluid and ionic balance.     

On the mechanism of lactate dehydrogenase release from skeletal muscle in relation to the control of cell volume

The mechanism of enzyme release from isolated skeletal muscle was illustrated by the study of the release of lactate dehydrogenase (LDH). In hypotonic media of different composition but of same tonicity the increase of LDH permeability was triggered at the same range of relative osmolality R (0.45 less than R less than 0.55), although the swelling in the respective media showed appreciable differences. The kinetics of muscle swelling showed that a deviation from the theoretically computed swelling curve to lower values of swelling was connected with an increased LDH permeability. The reduction of swelling was ATP- and Ca2+ and/or Mg2-dependent. It is concluded that swelling of cells generally precedes the leakage of soluble enzymes, and the cross-linking of filaments at the sarcoplasmic side of the sarcolemma under appropriate conditions can counteract swelling, thereby blebbing off the cell membrane from the filament meshwork. In the course of this process, sufficiently large membrane lesions are produced through which macromolecules may escape into the extracellular space.     

Permeability properties of the membrane of vesicular stomatitis virions

Observations of the light-scattering properties of several enveloped viruses indicate that virions (vesicular stomatitis, SV5 and influenza), in common with other membrane systems, are osmotically active, responding to NaCl gradients by swelling in hypo-osmolar solutions and shrinking in hyperosmolar solutions. The permeability barrier responsible for this osmotic response in vesicular stomatitis virions was modified both by protease treatment to remove the viral glycoprotein and by treatment with the polyene antibiotic filipin, an agent known to interact with cholesterol in liposomes and membranes. Filipin altered the kinetic and equilibrium permeability behavior of virions but the extent of leakage of osmotic shocking agent was less than that in lecithin/cholesterol and lecithin/ergosterol liposomes and in ergosterol-containing ciliary membranes. Negative-staining electron microscopy revealed that filipin treatment caused structural changes in the viral membrane. Intact virions exhibited appreciably larger responses to osmotic change than did protease-treated virus particles. Thus, the osmotic barrier in intact vesicular stomatitis virions may not be exclusively lipid in nature.     

Permeability properties of the membrane of vesicular stomatitis virions.

Observations of the light-scattering properties of several enveloped viruses indicate that virions (vesicular stomatitis, SV5 and influenza), in common with other membrane systems, are osmotically active, responding to NaCl gradients by swelling in hypo-osmolar solutions and shrinking in hyperosmolar solutions. The permeability barrier responsible for this osmotic response in vesicular stomatitis virions was modified both by protease treatment to remove the viral glycoprotein and by treatment with the polyene antibiotic filipin, an agent known to interact with cholesterol in liposomes and membranes. Filipin altered the kinetic and equilibrium permeability behavior of virions but the extent of leakage of osmotic shocking agent was less than that in lecithin/cholesterol and lecithin/ergosterol liposomes and in ergosterol-containing ciliary membranes. Negative-staining electron microscopy revealed that filipin treatment caused structural changes in the viral membrane. Intact virions exhibited appreciably larger responses to osmotic change than did protease-treated virus particles. Thus, the osmotic barrier in intact vesicular stomatitis virions may not be exclusively lipid in nature.     

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.     

Mechanism of stimulation of pinocytosis by trypan blue

Trypan blue at 50 microgram/ml stimulates the pinocytic uptake of 125I-labelled PVP, but not of colloidal 198Au or formaldehyde-denatured 125I-labelled bovine serum albumin, by the 17.5-day rat visceral yolk sac incubated in vitro. Neither Trypan blue nor a combination of the dye with 125I-labelled PVP stimulated the rate of pinocytosis of liquid by the tissue. Trypan blue itself was shown to enter the yolk-sac cells by adsorptive pinocytosis. It is proposed that an interaction between Trypan blue and 125I-labelled PVP enables the latter substrate to enter the cells adsorptively by so-called 'piggy-back' pinocytosis.     

The nature of the conductance increase induced by filipin in cholesterol-containing planar lipid bilayers

The effects of the polyene antibiotic filipin on the conductance and permeability of planar lipid bilayers were investigated under voltage-clamp conditions. The membrane conductance of lipid bilayers containing no cholesterol was not affected by filipin. In the presence of cholesterol containing lipid bilayers, filipin induced a 10(4)-10(5)-fold increase in transmembrane conductance. This conductance increase was dependent on the ionic species present in solution, decreasing in the following order: GCsCl greater than GNaAc greater than GKCl greater than GNaCl greater than CaCl2 greater than GNa2SO4 greater than GBaCl2 greater than GMgCl2. Reversal potential measurements in simple biionic conditions revealed the following relative permeability sequence: PK greater than PCl greater than PNa approximately Pac approximately PBa greater than PCs greater than PMg approximately PCa greater than Psulphate. The filipin-sterol mediated increase in membrane conductance was independent of the membrane potential. The increase in membrane current following a step alteration in membrane potential occurred instantaneously and had no dependence on the previous value of the holding membrane potential. We propose that the filipin-sterol complex forms ion channels in lipid membranes. These channels are found in a single configuration (open state) and select preferentially monovalent cations or anions over divalent ions. Our experimental results are discussed in relation to the effects of other polyene antibiotics on the membrane permeability, and also in relation to experimental problems previously reported with the use of filipin in planar lipid bilayers.     

Critical evaluation of techniques to detect and measure cell death--study in a model of UV radiation of the leukaemic cell line HL60.

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V-FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.     

Testicular organogenesis in the fetal calf: interstitial endocrine (Leydig) cell development

The development of the interstitial endocrine (Leydig) cells of the fetal testis in the calf is described and correlated with a swelling reaction of the gubernaculum and normal, prenatal descent of the testis. An hydroxysteroid dehydrogenase (HSD) procedure is used to determine the onset of functional activity for the interstitial endocrine cells (IEC). The NADH control procedure was strongly positive for the IECs at all ages investigated, indicating that these cells utilize the pyridine nucleotide as a coenzyme for oxireduction conversions. The 3 alpha- and 3 beta-HSD reactions were strongly positive and lightly positive, respectively, demonstrating that these cells contain the HSDs commonly utilized in the early steroidogenesis. TEM revealed structural evidence of this differentiating steroidogenic capability within IECs. During the period of the swelling reaction there is a functional IEC population, but there is no evidence presented by this study for a causal relationship of the gubernacular swelling reaction and subsequent normal descent of the testis into the scrotum.     

Effect of gamma radiation on resting B lymphocytes. I. Oxygen-dependent damage to the plasma membrane results in increased permeability and cell enlargement

Although the susceptibility of resting B lymphocytes to radiation-induced interphase death is well known, the mechanism by which this occurs is not understood. In this report, we use three measures of plasma membrane integrity (increase in cell volume, uptake of trypan blue, and release of 51Cr) to assess the effect of radiation on the resting B cell plasma membrane. The delivery of 500 to 1000 rad caused the majority of resting B cells to enlarge slightly, whereas 3000 rad caused virtually all of the cells to approximately double in size within 3 to 4 hr. Measurement of the release of 51Cr from resting B cells revealed a similar relationship between the dose of radiation and the loss of radioactive label. Trypan blue exclusion was also found to diminish as a function of radiation dose. An analysis of a variety of lymphoid cells suggested that sensitivity to the membrane damaging effects of gamma radiation was in the order of resting B cells greater than resting T cells greater than a long-term L3T4+ T cell clone greater than a B cell lymphoma. LPS-induced B cell blasts treated with 3000 rad were equivalent to 1000 rad-treated resting B cells. The effects of the gamma radiation could be ameliorated by excluding oxygen (a diradical molecule that can potentially enhance the generation and propagation of highly reactive free radicals) at the time of irradiation, or by adding the free radical scavenging agent cysteamine. These data are compatible with the hypothesis that gamma radiation results in damage to the plasma membrane of resting lymphocytes via the generation of highly reactive free radical species. This damage is reflected in a rapid increase in plasma membrane permeability and swelling of the cells, and may play a major role in causing interphase death.     

Phospholipid flippases and Sfk1 are essential for the retention of ergosterol in the plasma membrane

Sterols are important lipid components of the plasma membrane (PM) in eukaryotic cells, but it is unknown how the PM retains sterols at a high concentration. Phospholipids are asymmetrically distributed in the PM, and phospholipid flippases play an important role in generating this phospholipid asymmetry. Here, we provide evidence that phospholipid flippases are essential for retaining ergosterol in the PM of yeast. A mutant in three flippases, Dnf1-Lem3, Dnf2-Lem3, and Dnf3-Crf1, and a membrane protein, Sfk1, showed a severe growth defect. We recently identified Sfk1 as a PM protein involved in phospholipid asymmetry. The PM of this mutant showed high permeability and low density. Staining with the sterol probe filipin and the expression of a sterol biosensor revealed that ergosterol was not retained in the PM. Instead, ergosterol accumulated in an esterified form in lipid droplets. We propose that ergosterol is retained in the PM by the asymmetrical distribution of phospholipids and the action of Sfk1. Once phospholipid asymmetry is severely disrupted, sterols might be exposed on the cytoplasmic leaflet of the PM and actively transported to the endoplasmic reticulum by sterol transfer proteins.     

Caveolae and cholesterol distribution in vascular smooth muscle cells of different phenotypes.

Vascular smooth muscle cells (SMCs) grown in primary culture are converted from a contractile to a synthetic phenotype. This includes a marked morphological reorganization, with loss of myofilaments and formation of a large ER-Golgi complex. In addition, the number of cell surface caveolae is distinctly reduced and the handling of lipoprotein-derived cholesterol changed. Here we used filipin as a marker to study the distribution of cholesterol in SMCs by electron microscopy. In contractile cells, filipin-sterol complexes were preferentially found in caveolae and adjacent ER cisternae (present in both leaflets of the membranes). After exposure to LDL or cholesterol, labeling with filipin was increased both in membrane organelles and in the cytoplasm. In contrast, treatment with mevinolin (a cholesterol synthesis inhibitor) or beta-cyclodextrin (a molecule that extracts cholesterol from cells) decreased the reaction with filipin but did not affect the close relation between the ER and the cell surface. In synthetic cells, filipin-sterol complexes were diffusely spread in the plasma membrane and the strongest cytoplasmic reaction was noted in endosomes/lysosomes, both under normal conditions and after incubation with LDL or cholesterol. On the basis of the present findings, we propose a mechanism for direct exchange of cholesterol between the plasma membrane and the ER and more active in contractile than in synthetic SMCs.     

Influence of the culture oxygenation on the release of LDH by hybridoma cells

Summary The viability of hybridoma cells grown in reactors is investigated with two methods: the Trypan Blue staining of dead cells and the release of the cytoplasmic lactate dehydrogenase (LDH). Linear correlations are found between the number of counted dead cells and the measured LDH activity in the supernatant both when cells are grown with and without oxygen limitations. However the release of LDH is significantly higher for the oxygen limited culture (620 IU LDH / 10⁹ cells) than for the non-oxygen limited culture (270 IU LDH / 10⁹ cells).     

Counting human neural stem cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm(2) quadrant (0.1 microl) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in microl gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.     

Isolation and characterization of a polarized isolated hepatocyte preparation in the skate Raja erinacea

Hepatocytes of the small skate (Raja erinacea) were isolated by collagenase perfusion and evaluated by a variety of functional and morphologic criteria. Cell yield was 1.45 X 10(8) +/- 1.3 X 10(7) cells per isolation, and as long as 8 h after isolation 98% of the hepatocytes excluded Trypan blue and no leakage of lactate dehydrogenase (LDH) or cell associated potassium could be detected. Oxygen consumption averaged 1.6 +/- 0.5 nmol/min/mg cell protein, was not stimulated by 1 mM succinate, and also remained stable for up to 8 h following isolation. However, 2,4,-dinitrophenol (5 X 10(-5) M) produced a 55% increase in oxygen utilization while ouabain, (1 mM) or sodium removal decreased oxygen consumption by 31 +/- 6 and 33 +/- 7%, respectively, indicating that a significant portion of the cells energy utilization is coupled to the activity of plasma membrane Na+, K+-ATPase. Light microscopic studies showed that the individual hepatocytes had diameters of 28 +/- 5 microns and contained large lipid droplets. Electron microscopy revealed groups of three to five cells with normal ultrastructure and tight junctions and desmosomes surrounding a single bile canaliculus. These studies indicate that skate hepatocytes can be isolated in high yield that retain their structural polarity in the form of clusters of cells formed around a single bile canaliculus. These hepatocytes remain morphologically intact and metabolically stable for a prolonged period of time.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of lactate dehydrogenase (LDH) release kinetics for the evaluation of death and growth of mammalian cells in perfusion reactors

A general methodology is proposed to estimate the actual specific growth and death rate of mammalian cells in continuous perfusion reactors from the monitoring of the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) in the culture medium. The procedure is illustrated on a perfusion culture of human tumor kidney cells growing on microcarriers and producing prourokinase (PUK). The intracellular LDH content of living attached cells is checked to be constant during the culture. However, cells detached from the microcarriers, and counted dead because of the uptake of trypan blue, have only released part of their intracellular LDH. In the culture medium, LDH is relatively stable as the loss of activity does not exceed 5% per day. The time variation of the LDH concentration in the medium is used to calculate the total amount of lysed and actually produced cells in the reactors, hence, the actual specific rates of cell growth and death. It is thus found that the stationary phase observed after 400 h of perfusion culture is the result of equal growth and death rates, with a daily renewal of living cells on the microcarriers near 10%. Moreover, for the cell line tested, the production of PUK is associated with cellular growth.     

The localization of estrogen receptors on human osteoblasts

Attempts to identify estrogen target cells in bone by immunocytochemistry using antibodies to the receptor have proved to be controversial. The aim of this study was therefore to determine whether immunogold labeling can be used as a technique for the localization of estrogen receptors (ER) on a human osteoblast-like cell line. The aim was also to determine the distribution of ER on the cell surface by using a scanning electron microscope (SEM) and intracellularly by using a transmission electron microscope (TEM). Labeling of the cytoplasmic material was seen around areas that appeared to be a disrupted plasma membrane. No nuclear or perinuclear labeling could be detected. The conclusion can be made that SEM immunogold labeling combined with TEM can be regarded as a practical technique for localizing ER on human osteoblasts. This article clearly demonstrated that osteoblast-like cells do express ER at low levels and that, although cytoplasmic immunoreactivity could be detected, no, nuclear or perinuclear labeling was found.     

Assessment of the intensity of lymphocyte blast transformation in the crypts of the palatal tonsils by using DNA cytophotometry

Cell fractions isolated from lacunae of palatine tonsils with different functional activities were taken from 40 children 6-15 years of age. The cell fractions were examined using vital phase-contrast microscopy, light microscopy of Pappenheim-stained smears and the Feulgen cytophotometry. Lymphocytes with signs of immunological involvement were shown to constitute 95 per cent of the whole cell lacunar population of the active functional tonsils. 35 per cent of lymphocytes, being in different stages of blast transformation, were found. The most intense blast transformation was revealed in patients with tonsil inflammation. 5-13 per cent of lacunar nuclei with DNA content above the diploid level were found in these patients. Some multicellular islets accumulating small lymphocytes, lymphocytes at different stages of blast transformation, and macrophages were revealed to imitate the cooperation of immunocompetent cells. The data obtained can be helpful in the practical surgery of palatine tonsils.     

A comparative study of the inhibitory effect of trypan blue on mouse erythrocyte and C3 binding receptors of peripheral blood mononuclear cells from healthy donors and patients suffering from chronic lymphocytic leukaemia

Trypan blue has previously been shown to interact with the C3 receptor, but not with the Fc receptor. In the present work the effects of Trypan blue on mouse erythrocyte (ME) and C3 binding receptors of the peripheral blood mononuclear cells have been studied in healthy individuals and in patients with chronic lymphocytic leukemia (CLL). The resulting dose-response curves have been compared with each another. The results of statistical analysis indicate that there is no significant difference between the effects of trypan blue on the two receptors in healthy individuals. In contrast ME and C3 binding receptors differ significantly in trypan blue sensitivity in CLL patients. These data suggest that the two receptors are similar, or are situated on the cell membrane in such a way that the trypan blue bound to one of them inhibits the functioning of the other one too, but they are not the same. In the course of leukemic transformation, the trypan blue sensitivity of the ME binding receptor does not vary, whereas that of the C3 receptor is enhanced significantly.     

Intracellular uptake and alpha-amylase and lactate dehydrogenase releasing actions of the divalent cation ionophore A23187 in dissociated pancreatic acinar cells.

Intracellular uptake of A23187 and the increased release of amylase and lactate dehydrogenase (LDH) accompanying ionophore uptake was studied using dissociated acinar cells prepared from mouse pancreas. Easily detected changes in the fluorescence excitation spectrum of A23187 upon transfer of the ionophore from a Tris-buffered Ringer's to cell membranes were used to monitor A23187 uptake. Uptake was rapid in the absence of extracellular Ca2+ and Mg2+ (t1/2=1 min) and much slower in the presence of Ca2+ or Mg2+ (t1/2=20 min). Cell-associated ionophore was largely intracellular as indicated by fluorescence microscopy, lack of spectral sensitivity to changes in extracellular Ca2+ and Mg2+, and by equivalent interaction of ionophore with membranes of whole and sonicated cells. A23187 (10 micronm) increased amylase release 200% in the presence of extracellular Ca2+ and Mg2+. In the absence of Ca2+ (but in the presence of Mg2+) A23187 did not increase amylase release. A23187 (10 micronm) also produced Ca2+ -dependent cell damage, as judged by increased LDH release, increased permeability to trypan blue, and by disruption of cell morphology. The cell damaging and amylase releasing properties of A23187 were distinguished by their time course and dose-response relationship. A23187 (1 micronm) increased amylase release 140% without increasing LDH release or permeability to trypan blue.