The Use of Vital Dyes to Assess Embryonic Viability in the Hamster, Mesocricetus Auratus

Experiments were designed to assess the use of the vital dyes trypan blue and fluorescein diacetate as indicators of the viability of hamster ova and embryos. Exclusion of trypan blue and fluorescence with fluorescein diacetate showed high correlations with uptake of [³H]uridine by ova and further development of embryos in vitro. Ova killed by freezing and thawing incorporated [³H]uridine at background levels. Trypan blue exclusion and fluorescein diacetate uptake were highly correlated with each other (r = 0.99). Trypan blue and fluorescein diacetate serve as excellent indices of viability in ova and early embryos of hamsters.     

Fluorescein Diacetate Uptake to Determine the Viability of Human Fetal Cerebral Cortical Cells

We investigated the accuracy of fluorescein diacetate uptake as an indicator of the viability of human fetal cerebral cortical cells. Cortical cells from 16-26-week-old normal fetuses were studied. The cortices were dissociated mechanically with normal saline to make a suspension. Fluorescein diacetate uptake and trypan blue exclusion were compared as methods for examining cell viability. Our results show that fluorescein diacetate uptake is a simple and sensitive method for examining human fetal cortical cell viability.     

Inhibition of pinocytosis in rat yolk sac by trypan blue.

Day 17.5 yolk sacs from rats injected with partially denatured 125I-labeled bovine serum albumin (I-BSA) were cultured in vitro by a raft technique. The rates of release of [125I]iodotyrosine were similar in control yolk sacs and in yolk sacs from rats preinjected with trypan blue. Day 17.5 rat yolk sacs were also cultured in medium containing I-BSA. Following pinocytic uptake the substrate was degraded intracellularly and [135I]iodotyrosine released into the medium. Trypan blue, when present in the medium in concentrations above 100 mug/ml, inhibited pinocytosis of I-BSA and so decreased the rate of [125I]iodotyrosine production. Trypan blue similarly decreased the rate of pinocytic uptake of 125I-labeled polyvinylpyrrolidone. Pinocytic uptake of macromolecules was not decreased in yolk sacs from rats pretreated with trypan blue. The relevance of these results to the mechanism of teratogenic action of trypan blue is discussed. It is proposed that if trypan blue in teratogenic doses similarly inhibits pinocytosis by the yolk sac during the organogenetic period teratogenesis might result from a transient interruption in the flow of metabolites through the yolk sac to the embryo.     

Activation of Mouse Oocytes, Fertilization and Development of Mouse Embryos in Vitro after Staining with Trypan Blue or Fluorescein Diacetate

The influence of vital staining with trypan blue or fluorescein diacetate on the fertilization of mouse oocytes and the developmental potential of mouse embryos was assessed. Neither stain induced spontaneous activation in mouse oocytes, nor did they impair the in vitro development and implantation of mouse zygotes, two-cell embryos, stressed morulae or blastocysts. However, fertilization and subsequent development of mouse oocytes have been shown to be reduced by vital staining.     

Physiological effects of hypoxia and trypan blue in 17-day chick embryos.

Seventeen-day chick embryos were divided into 5 groups and treated as follows: (1) untreated, (2) yolk-sac injected with 0.1 ml of saturated trypan blue solution solution in sterile saline, (3) saline, (4) hypoxia, i.e., 10.5% oxygen, and (5) hypoxia plus trypan blue. After 5 h hypoxia-treated embryos had an increased mortality rate, severe hypoglycemia, reduced blood pH, elevated plasma potassium, and reduced CO2 content. Trypan blue treatment induced few deaths and few physiological imbalances. Hypoxia plus trypan blue was without synergistic effects and had effects that did not differ significantly from hypoxia alone. This lack of response of 17-day chick embryos to high doses of trypan blue may be related to a marked decline in oxygen consumption by the yolk at this age.     

Flow cytometric assessment of cell viability: a multifaceted analysis

Flow cytometry offers the possibility to simultaneously analyze, on a cell by cell basis, different parameters related to cell viability i.e. cell size, morphology and incorporation of dyes. Different types of analysis: light absorption of unstained/stained cells, forward angle light scattering (FALS), right angle light scattering (RALS) or both, cell fluorescence based on dye retention or dye exclusion (due to erythrosin B, ethidium bromide, fluorescein diacetate, rhodamine 123) were tested and compared, with the classical Trypan blue exclusion test, for their effectiveness in the determination of cell viability. Two types of cells in monolayer cultures (L929, SIRC) and a freshly isolated suspension of mouse splenocytes were used. For each dye, the optimal dose, incubation time and conditions for analysis were determined. Viability indications by different techniques for the three type of cell line and their reliability as compared with Trypan blue were analyzed.     

Studies on the mechanism of trypan blue teratogenicity in the rat developing in vivo and in vitro

Trypan blue is a potent teratogen in vivo and in vitro in the rat. Many of the abnormalities produced by trypan blue--including swollen neural tube and pericardium, subectodermal blisters, hematomas, and generalized edema--may result from altered fluid balance in and around the embryo. The present study demonstrates relationships between changes in the fluid environment around the embryo and appearance of anomalies. Rat embryos were exposed in utero or in vitro to trypan blue during the early period of organogenesis. Both exposures resulted in defects that are typical of trypan blue treatment. Osmolality of exocoelomic fluid (ECF) was measured on gestation day 10 in vivo and day 12 in vitro, both after 48 hr of exposure to trypan blue. In both cases ECF osmolality was significantly lower than controls. This was correlated with the presence of edema-related anomalies in the embryo. On gestation day 11 in vivo, three days after maternal injection of trypan blue, ECF osmolalities were significantly higher than controls; however, there was tremendous variability in this parameter in day 11 treated embryos, and some had ECF osmolalities below the control range. Increased frequency of abnormalities was correlated with abnormal ECF osmolality, below and above the control range. Trypan blue probably exerts its teratogenic effects by disturbing the function of the visceral yolk sac. The movements of an amino acid and a monosaccharide across the visceral yolk sac were measured on gestation day 12 embryos in vitro. This aspect of yolk sac function was not altered by trypan blue exposure. Ultrastructure of the visceral yolk sac was observed after trypan blue exposure in vivo and in vitro. Endodermal cells in trypan blue-treated yolk sacs contained fewer large, electron dense lysosomes than controls. These were replaced by numerous small vacuoles, which may contain trypan blue. Trypan blue causes osmotic changes in the rat embryo in vivo and in vitro. These changes are correlated with embryonic malformations. Alterations in yolk sac ultrastructure indicate that trypan blue affects the function of this membrane.     

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.     

Stimulation of uridine phosphorylation in primary cultures of Xenopus laevis hepatocytes by estradiol-17 beta

The effects of estrogen on the uridine uptake into cells were examined in primary cultures of liver parenchymal cells from Xenopus laevis. The total uptake of [3H]uridine into the estrogen-treated cells and its incorporation into RNA were about 1.5 times higher than the values for control cells. The uptake of [3H]adenosine and its incorporation into RNA were not affected by estrogen. An experiment in which liver parenchymal cells were double labeled with [3H]uridine and [3H]adenosine showed that estrogen elevated the specific radioactivity of the UTP pool 1.4-fold the value found for the control cells, but that of the ATP pool was not altered by estrogen. Short term labeling revealed that estrogen did not significantly alter the rate of the initial uptake of [3H]uridine into the cells, but it did stimulate [3H]uridine phosphorylation about 1.7-fold. Uridine kinase activity measured in cell-free extracts of hepatocytes treated with estrogen had a value 1.6 times that of the control cells. These data indicate that the stimulation of [3H]uridine uptake and phosphorylation in Xenopus laevis hepatocytes in the presence of estrogen is caused by the enhancement of uridine kinase activity.     

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     

Comparison of overall rates of RNA synthesis in implanting and delayed implanting mouse blastocysts in vitro

Implanting and delayed implanting mouse embryos were incubatedin vitro with [³H]uridine for 2–24 hr. The size and specific activity of the [³H]UTP pools were determined by means of a double isotope technique using copolymer synthesis with the [³H]UTP in the embryos, exogenous [¹⁴C]ATP, andE. coli RNA polymerase. Using the rate of incorporation of [³H]uridine into acid-insoluble material and the specific activity of the [³H]UTP pools, it was possible to calculate the overall rate of incorporation of uridine into RNA by the embryos. In implanting embryos it was constant for 24 hr. In contrast, the initial rate of uridine incorporation by the delayed implanting embryos was only 31% of that in implanting embryos (i.e., per cell); this increased steadily during the incubation period, reaching 81% of the rate in implanting embryos after 24 hr. This activation of RNA synthesis by delayed implanting embryosin vitro occurred in the absence of any uterine stimulatory factors. Further, it was shown that although 10% mouse serum would support trophoblastic outgrowthin vitro, it did not influence uptake, distribution of label into nucleotides, or rate of uridine incorporation into RNA in either implanting or delayed implanting embryos. Therefore, it is suggested that if depression and activation of metabolic activity in blastocysts are part of the mechanims of delayed implantation, and if trophoblast outgrowthin vitro is analogous to the process of implantationin vivo, then these two aspects of embryo activation are under different controls.     

Autoradiographic labeling of spinal cord neurons with high affinity choline uptake in cell culture

Embryonic chick spinal cord neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline. We find that, in addition to acetylcholine synthesis, the accumulated choline is used for the synthesis of metabolites such as lipids that are retained in part by conventional fixation techniques. As a result autoradiographic methods can be used to identify the cells that have the uptake mechanism in spinal cord cultures. About 60% of the neurons are labeled by [³H]choline uptake in cultures prepared with spinal cord cells from 4-day-old embryos, and about 40% are labeled in cultures prepared with cord cells from 7-day-old embryos. Neurons that innervate skeletal myotubes in spinal cord-myotube cultures are consistently labeled by [³H]choline uptake. Neurons unlabeled by the procedure are viable: they exclude the dye trypan blue and accumulate ¹⁴C-amino acids for protein synthesis. Most of the neurons unlabeled by [³H]choline uptake can instead be labeled by uptake of γ-[³H]aminobutyric acid, and vice versa. These results suggest that high affinity choline uptake can be used to label cholinergic neurons in cell culture, and that at least some populations of noncholinergic neurons are not labeled by the procedure. It cannot yet be concluded, however, that all labeled neurons are cholinergic since more labeled neurons are obtained per cord than would be expected from the number of neurons making up identified cholinergic populations in vivo. A three- to fourfold increase in the amount of high affinity choline uptake is observed between Days 3 and 15 in culture for spinal cord cells obtained from 4-day-old embryos. The number of [³H]choline-labeled neurons in such cultures decreases slightly during the same period, suggesting that the increase in uptake reflects neuronal growth or development rather than an increase in population size. Both the magnitude of the uptake and the number of [³H]choline-labeled neurons are the same for spinal cord cells grown with and without skeletal myotubes.     

Initiation of DNA synthesis in murine B cells is independent of early changes in the cytosolic free calcium

The relationship between changes in the intracellular free Ca2+ concentration, [Ca2+]i, and the initiation of proliferation of murine B cells after the addition of mitogens and activators was studied. The effects of lipopolysaccharide (LPS), 12-O-tetradecanoyl phorbol-13-acetate (TPA), rabbit IgG antimouse Fab (IgG RAM Fab), and its F(ab')2 fragment (F(ab')2 anti-Fab) on the [Ca2+]i were measured using the fluorescent calcium indicator Fura-2. In parallel experiments, DNA and/or RNA synthesis were measured by assaying [3H]thymidine and/or [3H]uridine uptake. LPS stimulated a 20-120 X increase in the [3H]thymidine uptake, and a 3-7 X increase in [3H]uridine uptake without inducing any change in the [Ca2+]i. TPA induced a marginal increase in [3H]thymidine and [3H]uridine uptake, without effecting any change in the [Ca2+]i. In contrast, low doses of IgG RAM Fab produced a triphasic change in the [Ca2+]i, but had no effect on the [3H]thymidine or [3H]uridine uptake, even at much higher concentrations. Similarly, low doses of the F(ab')2 fragment induced sizable increases in the [Ca2+]i without affecting the [3H]nucleoside uptake. However, higher concentrations of F(ab')2 anti Fab increased the [3H]thymidine uptake and [3H]uridine uptake, while also increasing the [Ca2+]i. Significantly, pretreating the cells with TPA for 3 min virtually abolished the [Ca2+]i increase induced by IgG RAM Fab while simultaneously potentiating an increase in the IgG RAM Fab-induced [3H]thymidine uptake 85-fold. In the presence of TPA, IgG RAM Fab also induced a 2- to 30-fold increase in [3H]uridine uptake. Similarly, TPA virtually abolished the [Ca2+]i increase induced by the F(ab')2 anti-Fab fragment, yet it stimulated a F(ab')2 anti-Fab-induced uptake of [3H]thymidine and [3H]uridine by 120 and 10 times, respectively.     

The uptake in vitro of dyes, monosaccharides and amino acids by the filarial worm Brugia pahangi.

The uptake in vitro of various substances by Brugia pahangi was investigated using infective larvae obtained from Aedes aegypti and worms removed from Meriones unguiculatus at 2, 3, 10, 20 and 90 days post-infection. Worms incubated in growth medium 199 containing 1% Trypan blue possessed demonstrable dye in the oral orifice, the anterior oesophageal lumen and the external openings of the vulva and the cloaca or anus but the dye was not found in the gut lumen even after incubation for 24 h. No uptake of ferritin particles into the intestine of the worms was found and no fluorescence could be demonstrated in the gut lumen of worms incubated in medium containing 50% (v/v) fluorescein isothiocyanate-conjugated calf serum for up to 24 h. Trypan blue uptake by the gut of Aspiculuris tetraptera was clearly observed after incubation for several hours. The uptake of D-glucose and L-leucine by B. pahangi was demonstrated using autoradiographic and scintillation counting techniques and incorporation into worm tissues was detected. Glucose was found to be readily incorporated in the apical, glycogen-rich areas of the myocytes of worms of all ages studied and in the uterine epithelium of the adult female. In contrast, a lower incorporation of D-glucose was found in the eggs, embryos and vas deferens and especially in the gut. The incorporation of L-leucine occurred throughout the tissue of the worms during a 30 min incubation. Labelling was also located over the surface of the cuticle of the worms, when incubated for a period of 15 to 60 min in L-[H]leucine. Scintillation counting techniques demonstrated that there was no uptake of 14C-labelled L-glucose or sucrose by B. pahangi. The data presented on the uptake in vitro of nutrients or other compounds by infective larvae and adult stages of B. pahangi did not demonstrate an intestinal route of uptake but indicated that the transcuticular route of uptake may be employed.     

Cytidine is a novel substrate for wild-type concentrative nucleoside transporter 2

Nucleoside transporter (NT) plays key roles in the physiology of nucleosides and the pharmacology of its analogues in mammals. We previously cloned Na+/nucleoside cotransporter CNT2 from mouse M5076 ovarian sarcoma cells, the peptide encoded by it differing from that by the previously reported mouse CNT2 in five substitutions, and observed that the transporter can take up cytidine, like CNT1 and CNT3. In the present study, we examined which of the two aforementioned CNT2 is the normal one, and whether or not cytidine is transported via the previously reported CNT2. The peptide encoded by CNT2 derived from mouse intestine, liver, spleen, and ovary was identical to that previously reported. The uptake of [3H]cytidine, but not [3H]thymidine, by Cos-7 cells transfected with CNT2 cDNA obtained from mouse intestine was much greater than that by mock cells, as in the case of [3H]uridine, a typical substrate of NT. [3H]Cytidine and [3H]uridine were taken up via CNT2, in temperature-, extracellular Na+-, and substrate concentration-dependent manners. The uptake of [3H]cytidine and [3H]uridine mediated by CNT2 was significantly inhibited by the variety of nucleosides used in this study, except for thymidine, and inhibition of the [3H]uridine uptake by cytidine was competitive. The [3H]uridine uptake via CNT2 was significantly decreased by the addition of cytarabin or gemcitabine, antimetabolites of cytidine analogue. These results indicated that the previously reported mouse CNT2 is the wild-type one, and cytidine is transported mediated by the same recognition site on the CNT2 with uridine, and furthermore, cytidine analogues may be substrates for the transporter.     

Two-dimensional fluorescence spectroscopy – a new tool for the determination of plant cell viability

 Two-dimensional fluorescence spectroscopy (2D-FS) has been used as a new method for determining the viability of tobacco cells (Nicotiana tabacum L.). Both horizontal beam geometry and a vertical set-up achieved with bifurcated fibres were tested. The latter arrangement enabled us to avoid the negative effect of cell sedimentation. Incubation of a tobacco BY-2 cell suspension with dimethylsulfoxide (DMSO) (0–10% v/v) resulted in cell samples differing in their viability – from fully viable (0–2% DMSO) to totally non-viable (8–10%DMSO). The validity of determining viability by means of measuring cell esterase activity by 2D-FS using fluorescein diacetate as a fluorogenic substrate was verified by comparison with microscopic evaluation of fluorescein fluorescence as well as with the routinely adopted trypan blue exclusion test. Received: 6 June 2000 / Revision received: 9 October 2000 / Accepted: 9 October 2000     

Persistence of embryonic RNA synthesis during facultative delayed implantation in the mouse.

The status of embryonic RNA synthesis during facultative delayed implantation in the mouse has been examined by radiolabeling in vitro and in utero, and by assay for endogenous RNA polymerase activity. Under conditions that do not activate delayed blastocysts in utero, embryos were shown to be able to transport and incorporate [³H]uridine into RNA as early as 5 min after intralumenal instillation of label on Day 5 of delay. Assay for endogenous RNA polymerase demonstrated functioning enzyme(s) in blastocysts on Day 5 of delayed implantation. Rates of incorporation of label in vitro under nonactivating conditions indicated a reduction, from normal Day 5 blastocyst levels, of 52% on Day 2 and 36% on Day 5 of delay. Relative rates of uptake of [³H]uridine by blastocysts on Day 5 of delay were reduced by approximately 60% from rates observed in predelay embryos on Day 5 of pregnancy. Estrogen-induced activation of embryos in utero was not associated with an increased relative rate of ³H]uridine uptake or incorporation during the first 24 hr following activation on Day 5 of delay. The findings demonstrate that RNA synthesis persists in the mouse blastocyst during delayed implantation, although at a somewhat reduced level. Implications of these results relevant to the maternal regulation of embryonic growth and implantation are discussed.     

An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide

A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.     

Amphetamines reduce embryonic size and produce caudal hematomas during early chick morphogenesis

Experiments were designed to study some of the similarities and differences in the effects of amphetamines and trypan blue on early chick morphogenesis. Both dextroamphetamine sulfate (0.5 mg/egg) and methamphetamine hydrochloride (1.0 mg/egg) were capable of inducing, in 3-day chick embryos, caudal hematomas which were similar in appearance and location to those routinely observed following treatment with trypan blue. It was found, too, that both dextroamphetamine and methamphetamine treated embryos frequently exhibited a significant decrease in crown rump length and cross-sectional area of the notochord, neural tube, dorsal aortae and whole body section, when compared with unopened or saline injected controls. Trypan blue treated embryos had only a rare decrease or increase in the size of structures when compared to either control group. These findings suggest that the amphetamines have an ability to decrease or retard embryonic growth in the chick.