Evaluation of a porcine lens and fluorescence assay approach for in vitro ocular toxicological investigations

Cell biology, as monitored with the fluorescent indicator dyes Alamar Blue and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), and lens optical quality, as measured with an in vitro scanning laser system, have been used to evaluate in vitro the condition of porcine lenses after being placed in a culture medium. The measurements, beginning from week one of culture, were compared statistically. Optical quality and cellular viability, as measured with either dye, were unchanged in lenses that had been maintained for 6 weeks in modified M199 medium. Some lenses were treated with 0.152J/cm(2) UVB radiation, and a decline was observed after 48 hours in both optical and metabolic capabilities, as indicated by a decreased capacity of the lenses to reduce Alamar Blue. The measurements with CFDA-AM did not show complete concordance with the other indicators of lens health after UV treatment, making this dye less reliable as applied currently to lens cultures. Overall, the findings suggest that porcine lenses can be maintained for weeks in culture, and that their condition can be evaluated quantitatively by assays that probe cellular functions and optical properties. Such a system should prove valuable for in vitro ocular pharmacotoxicological research.     

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.     

A versatile assay for the accurate,time-resolved determination of cellular viability

A convenient and versatile method for the accurate, time-resolved determination of cellular viability has been developed. The conventional viability indicator fluorescein diacetate (FDA), which is converted to the fluorescent compound fluorescein in living cells, was employed as a viability probe. Fluorescence emission from cells was measured using a spectrofluorimeter equipped with a magnetic stirrer. Using this assay cell suspensions exhibiting densities in the range 0.5 x 10(5) to 2.0 x 10(5) cells displayed a linear response when FDA concentrations less than 12 micro M were employed. To calibrate the method, viability standards were elaborated using different proportions of living and dead cells, and a correlation coefficient for the viability of tobacco BY-2 suspensions was calculated as 0.998. This viability assay was also found to be applicable to Chlamydomonas reinhardtii and Arabidopsis thaliana cultured cells. Using this cell viability assay, kinetic analyses of cell death could be performed. Using the proteinaceous elicitor from Phytophthora cryptogea, cryptogein, to induce cell death in tobacco cell suspensions, values for the maximum velocity of death induction rate (V(max)) and the LD50 (half-maximal velocity or k(1/2)) were calculated as 17.2 (% death/h) and 65 nM, respectively.     

Examination of mineralized nodule formation in living osteoblastic cultures using fluorescent dyes

Detecting the formation of mineralized nodules in osteogenic cell culture provides a means of assessing mature osteoblast cell function and the status of culture. In the present study, to continuously monitor the formation of mineralized nodules during the entire culture period, different concentrations of two fluorescent dyes (xylenol orange and calcein blue) were evaluated for their ability to specifically label calcified areas and their toxicity to cells in osteogenic cultures. Results showed that 20 microM xylenol orange and 30 microM calcein blue gave rise to distinct fluorescent staining for mineralized nodules, which were correlated exactly with von Kossa and alizarin red S staining at the same locations in cultures. In the assessment of toxicity, both dyes at the aforementioned concentrations did not alter cell viability or change the total DNA content in cultures. To demonstrate the advantage of using these fluorochromes to monitor mineralized nodules formation, consecutive fluorescent images of each staining were recorded at the same location of individual culture over the entire duration. The result indicates that both xylenol orange and calcein blue can provide contrasting fluorescent staining to continuously monitor mineralized nodules formation in living osteogenic cell cultures without deleterious effects.     

Culture conditions affect induction of vitellogenin synthesis by estradiol-17 beta in primary cultures of tilapia hepatocytes

In vitro synthesis of vitellogenin (VTG), a female-specific protein, after estradiol-17 beta (E(2)) treatment was compared among different culture conditions using the hepatocytes of tilapia, Oreochromis mossambicus. VTG was measured by enzyme-linked immunosorbent assay. Comparison of Leibovitz's L-15 medium (L-15), Williams' medium E (WE) and Medium 199 (M199), which have been used for hepatocyte cultures in certain teleost fishes, showed that monolayer formation of the hepatocytes on the plate in WE and M199 was faster than in L-15 at the beginning of the culture. Morphological differences in the hepatocytes among the culture media were not evident by 96 h after culture. VTG synthesis in L-15 after E(2) treatment was higher than in WE and M199. A concentration of NaHCO(3) at 5 mM in L-15 resulted in faster monolayer formation of the cells and higher VTG synthesis than at 0 and 23 mM. Primary culture of the tilapia hepatocytes at 28 degrees C showed higher synthesis of VTG than at 23 and 33 degrees C. These results suggest that nutritional requirements are vitally different among species, and there are optimal ranges in the pH and the temperature in cultured hepatocytes.     

MTT法用于药物对L2细胞毒性的实验研究

目的：评价噻唑蓝（MTT）法检测药物对细胞的毒性作用的可靠性。方法：大鼠肺泡上皮L2细胞以叔丁基对苯二酚（TBHQ）10．100μM,BsO以1-10mM分别处理,用MTT法检测细胞活性、JC-1（5,5’,6,6’-四氯．1,1’,3,3’-四乙基苯并咪唑羰花青碘化物）荧光染料法检测细胞线粒体电位改变、台盼蓝排斥实验检测细胞死亡率,分析各指标的情况。结果：在处理剂量范围,MTT法检测到的光密度（OD）值未能达到一般判断的半数抑制浓度（ic50）水平,最高抑制率仅达到30％左右;台盼蓝排斥试验检测数据表明TBHQ的LC50值为50μM,丁硫氨酸亚砜胺（BSO）为5mM;利用JC-1荧光染料判断的半数凋亡剂量分别为50μM和7mM。结论：MTT法作为最常采用的细胞生长抑制检测手段,但在某些特定实验中可能不能客观地反映细胞的活性,建议多种方法结合进行评价。     

A novel technique for viable cell determinations

We have developed a simple method to determine cell viability using two fluorescent dyes, Hoechst 33258 and acridine orange. When these dyes are used in combination, dead cells fluoresce brilliant blue and live cells fluoresce green. This method works over a range of dye concentrations (Hoechst 33258, 0.25-2 micrograms/ml; acridine orange, 1-5.0 micrograms/ml) and the fluorescence spectra of the two dyes are such that only one set of filters is required to visualize the effects of both dyes simultaneously. It is insensitive to a wide range of exogenous serum concentrations and is read with greater uniformity by different observers.     

Rapid assessment of islet viability with acridine orange and propidium iodide

Summary A simple, rapid method for estimating the viability of isolated islets of Langerhans with fluorescent dyes is described. Low concentrations of acridine orange and propidium iodide (AO/PI) were used to visualize living and dead islet cells simultaneously. AO/PI-stained islets can be divided into three distinct groups. Group A islets fluoresce green, contain insulin, and have normal ultrastructure; group C islets fluoresce primarily red, contain little or no insulin, and have cells with disrupted cellular membranes. Group B islets fluoresce red, green, and yellow. The yellow color is due to the addition of two primary colors from the superimposed red and green fluorescing cells. In this assay, the interpretation that red islet cells are dead and green islet cells are alive was confirmed by sequentially staining single islet cells with AO/PI and trypan blue. The observation that red islets are dead was confirmed by heat-killing, enzymatically damaging, treating with ethanol, or depriving islets of nutrients and observing the red fluorescence. This assay should be useful in studies where the assessment of islet viability is essential. Preliminary reports of this work were presented at two meetings and were published in abstract form (24,25). This research was supported in part by the National Institutes of Health, Bethesda, MD, grant DK 18115.     

Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG2 cells

Ultrasound-targeted microbubble destruction had been employed in gene delivery and promised great potential. Liver has unique features that make it attractive for gene therapy. However, it poses formidable obstacles to hepatocyte-specific gene delivery. This study was designed to test the efficiency of therapeutic gene transfer and expression mediated by ultrasound/microbubble strategy in HepG2 cell line. Air-filled albumin microbubbles were prepared and mixed with plasmid DNA encoding low density lipoprotein receptor (LDLR) and green fluorescent protein. The mixture of the DNA and microbubbles was administer to cultured HepG2 cells under variable ultrasound conditions. Transfection rate of the transferred gene and cell viability were assessed by FACS analysis, confocal laser scanning microscopy, Western blot analysis and Trypan blue staining. The result demonstrated that microbubbles with ultrasound irradiation can significantly elevate exogenous LDLR gene expression and the expressed LDLRs were functional and active to uptake their ligands. We conclude that ultrasound-targeted microbubble destruction has the potential to promote safe and efficient LDLR gene transfer into hepatocytes. With further refinement, it may represent an effective nonviral avenue of gene therapy for liver-involved genetic diseases.     

A Novel Technique for Viable Cell Determinations

We have developed a simple method to determine cell viability using two fluorescent dyes, Hoechst 33258 and acridine orange. When these dyes are used in combination, dead cells fluoresce brilliant blue and live cells fluoresce green. This method works over a range of dye concentrations (Hoechst 33258, 0.25-2 μg/ml; acridine orange, 1-5.0 μg/ml) and the fluorescence spectra of the two dyes are such that only one set of filters is required to visualize the effects of both dyes simultaneously. It is insensitive to a wide range of exogenous serum concentrations and is read with greater uniformity by different observers.     

Estimating viability of plant protoplasts using double and single staining

Summary The utility of numerous dyes for determining the viability of barley (Hordeum vulgare L. cv. Himalaya) aleurone protoplasts was studied. Protoplasts isolated from the barley aleurone layer synthesize and secrete -amylase isozymes in response to treatment with gibberellic acid (GA) and Ca²⁺. These cells also undergo dramatic morphological changes which eventually result in cell death. To monitor the viability of protoplasts during incubation in GA and Ca²⁺, several types of fluorescent and nonfluorescent dyes were tested. Evans blue and methylene blue were selected as nonfluorescent dyes. Living cells exclude Evans blue, but dead cells and cell debris stain blue. Both living and dead cells take up methylene blue, but living cells reduce the dye to its colorless form whereas dead cells and cell debris stain blue. The relatively low extinction coefficient of these dyes sometimes makes it difficult to distinguish blue-stained cells against a background of blue dye. Several types of fluorescent dyes were tested for their ability to differentially stain dead or living cells. Tinopal CBS-X, for example, stains only dead cells, and its high extinction coefficient allows its ultraviolet fluorescence to be recorded even when preparations are simultaneously illuminated with visible light. To double-stain protoplasts, the most effective stain was a combination of fluorescein diacetate (FDA) and propidium iodide (PI). By employing a double-exposure method to record the fluorescence from cells stained with both FDA and PI, dead and living cells could be distinguished on the basis of fluorochromasia.     

Participation of high-density lipoprotein in vitellogenesis in Japanese eel hepatocytes

To investigate effect of estradiol-17β (E₂) treatment in vivo on binding of eel hepatocytes to HDL, we developed hepatocytes binding assay. When hepatocytes were incubated with 200 times excess of eel HDL, the binding of hepatocytes to HDL precoated on wells was inhibited competitively. This indicates that eel hepatocytes bound specifically to HDL. E₂ treatment in vivo induced vitellogenin (VTG) synthesis. Hepatocytes prepared from the same E₂ treatment eel showed 3-fold higher ability of binding to HDL compared to hepatocytes prepared from ells without E₂ treatment. We also examined effects of E₂ and HDL on VTG induction in cultured hepatocytes. VTG, determined by enzyme-linked immunosorbent assay (ELISA), induction was about two-times higher in the presence of both 10⁻⁵ M of E₂ and 400 μg of HDL than in the presence of 10⁻⁵ M E₂ alone. At concentrations below 10⁻⁶ M E₂, VTG was not induced in the presence or absence of HDL. By SDS–PAGE and immunoblotting, VTG was detected only in the presence of both 10⁻⁵ M of E₂ and HDL. Our findings strongly support the idea that HDL correlates with vitellogenesis in eel liver.     

Green fluorescent protein: A novel viability assay for cryobiological applications

Assessment of tissue viability following the application of a freezing protocol is challenging due to the paucity of viability assays that can be used dynamically, in situ. Cells transfected with a green fluorescent protein (GFP) vector actively produce GFP, which is retained intracellularly. Because of its constitutive and heritable expression, GFP fluorescence of transfected cells may have significant utility as a viability assay for cells within tissues. As a first step toward application to tissues, this work seeks to establish the validity of this GFP-based assay in cell suspensions by comparing the results to other accepted measures of viability. To the authors' knowledge, this is the first use of GFP in cryobiology applications. Intracellular GFP fluorescence was evaluated following slow freezing. Nontransfected and GFP-transfected rat 3230 adenocarcinoma (R3230AC) cells were frozen at 1 degrees C/min to minimum temperatures between -5 and -30 degrees C and then immediately thawed in a 37 degrees C water bath. Samples were assayed using the common viability indicators trypan blue and ethidium bromide (EtBr). A regression analysis of recovery measured with the GFP assay as a function of recovery measured with a trypan blue assay gave a correlation coefficient of 0.97. A similar correlation coefficient, 0.95, was determined for recovery assessed by the GFP assay as a function of recovery measured by an EtBr assay. Nontransfected and GFP-transfected cells responded similarly to slow freezing, indicating that GFP transfection did not significantly alter the response of cells to typical freezing conditions. The excellent correlation of GFP assay results with those of two common viability assays suggests that the GFP-based assay is valid for cells and that further development of a tissue viability assay based on transfection is appropriate.     

A test of granulocyte membrane integrity and phagocytic function.

An assay of granulocyte viability has been developed which yields information about rwo important cell parameters, cell membrane integrity and phagocytic activity. The assay is based on the fact that only live cells can accumulate fluorescein, which is enzymatically split from the nonfluorescent substrate fluorescein diacetate. Dead cells, on the other hand, become permeable to the fluorescent red dye ethidium bromide. When cells are exposed first to opsonized zymosan particles, which they can phagocytize, then to a combination of these fluorescent dyes, one can distinguish microscopically between dead cells with fluorescent red nuclei, live cells which fluoresce green, and live cells with phagocytic function which are swollen with the pink zymosan particles in a green fluorescing cytoplasm. This assay takes 20--30 min and can be used to distinguish different degrees of cellular damage after cryopreservation.     

Development of a Robust Flow Cytometric Assay for Determining Numbers of Viable Bacteria

Several fluorescent probes were evaluated as indicators of bacterial viability by flow cytometry. The probes monitor a number of biological factors that are altered during loss of viability. The factors include alterations in membrane permeability, monitored by using fluorogenic substrates and fluorescent intercalating dyes such as propidium iodide, and changes in membrane potential, monitored by using fluorescent cationic and anionic potential-sensitive probes. Of the fluorescent reagents examined, the fluorescent anionic membrane potential probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(inf4)(3)] proved the best candidate for use as a general robust viability marker and is a promising choice for use in high-throughput assays. With this probe, live and dead cells within a population can be identified and counted 10 min after sampling. There was a close correlation between viable counts determined by flow cytometry and by standard CFU assays for samples of untreated cells. The results indicate that flow cytometry is a sensitive analytical technique that can rapidly monitor physiological changes of individual microorganisms as a result of external perturbations. The membrane potential probe DiBAC(inf4)(3) provided a robust flow cytometric indicator for bacterial cell viability.     

Cytotoxicity as measured by trypan blue as a potentially confounding variable in the in vitro alkaline elution/rat hepatocyte assay

Rat hepatocytes treated in vitro with A2RA, an angiotensin II receptor antagonist, displayed increased level of DNA-strand breaks as determined by alkaline elution, without an appreciable increase in cytotoxicity as determined by a trypan blue dye exclusion assay at harvest. The alkaline elution profile appeared to have two components: a rapidly eluting component detected in the first fraction collected (often associated with DNA from dead or dying cells), followed by a more slowly eluting component detected in the subsequent fractions. Further analysis of hepatocytes treated with A2RA by pulsed-field gel electrophoresis and neutral elution revealed significant levels of DNA double-strand breaks. Electron microscopy (EM) showed pronounced damage to mitochondria; although cell blebbing was seen using both EM and light microscopy, the plasma and nuclear membranes appeared intact when examined by EM. Cellular ATP levels decreased precipitously with increasing doses of A2RA, falling to less than 10% of control values at a dose of 0.213 mM A2RA, a concentration showing 100% relative viability by trypan blue at harvest. Thus, whereas in our experience trypan blue dye exclusion accurately reflects cytotoxicity induced by the majority of test agents, in this rather unusual case, trypan blue did not accurately reflect compound-induced cytotoxicity at harvest since there was no concurrent loss of membrane integrity. However, when hepatocytes treated with A2RA were incubated for either 3 h or 20 h in the absence of compound, a sharp, dose-dependent decline in viability was observed using trypan blue dye exclusion. Together with the initial, dose-dependent drop in the alkaline elution curve, these data suggest that the observed DNA double-strand breaks arose as a consequences of endonucleolytic DNA degradation associated with cytotoxicity, rather than by a direct compound-DNA interaction. Since DNA double-strand breaks behave under alkaline denaturing conditions as two single-strand breaks and can therefore produce increases in the alkaline-elution slope values, a necessary criteria for a valid positive result in this assay is that cytotoxicity by trypan blue dye exclusion will not be greater than 30%. Our data, however, indicate that interpretation of the elution assay as a test for genotoxicity can still be confounded by the failure of the trypan blue dye exclusion assay to reflect cytotoxicity in the unusual instance when there is no concurrent, immediate loss of membrane integrity.     

Effects of estradiol-17beta treatment on in vitro and in vivo synthesis of two distinct vitellogenins in tilapia.

Two distinct vitellogenins (VTG) were purified from the blood of estradiol-17beta (E(2))-injected tilapia, Oreochromis mossambicus. Enzyme-linked immunosorbent assays (ELISA) of each VTG were developed to examine effects of E(2) treatment on induction of VTG synthesis in the primarily cultured tilapia hepatocytes. Two VTG molecules (VTG210 and VTG140) had apparent molecular masses of 370 and 220 kDa by gel filtration and 210 and 140 kDa by SDS-PAGE, respectively. Western blot analyses showed that antibodies raised against the purified VTG210 and VTG140 reacted only with each protein band. Furthermore, ELISA for each VTG was specific for target VTG. When E(2) was added into the media of primarily cultured tilapia hepatocytes, VTG210 and VTG140 were both detected from E(2) concentrations of 1x10(-7) M and 5x10(-7) M, respectively. Time course experiments showed that there was a difference in the detection time of VTG210 and VTG140 after the hormone treatment. Although the injection of different E(2) doses induced both VTGs in the plasma of male tilapia, the concentration of VTG210 was nearly five to eight times higher than that of VTG140. These results suggest that E(2) is a direct inducer of both VTGs in the tilapia hepatocytes in vitro and in vivo, and that there is difference in the hormone response in inducing the VTGs in the tilapia hepatocytes.     

Multiplexed labeling system for high-throughput cell sorting

Flow cytometry and fluorescence activated cell sorting techniques were designed to realize configurable classification and separation of target cells. A number of cell phenotypes with different functionalities have recently been revealed. Before simultaneous selective capture of cells, it is desirable to label different samples with the corresponding dyes in a multiplexing manner to allow for a single analysis. However, few methods to obtain multiple fluorescent colors for various cell types have been developed. Even when restricted laser sources are employed, a small number of color codes can be expressed simultaneously. In this study, we demonstrate the ability to manifest DNA nanostructure-based multifluorescent colors formed by a complex of dyes. Highly precise self-assembly of fluorescent dye-conjugated oligonucleotides gives anisotropic DNA nanostructures, Y- and tree-shaped DNA (Y-DNA and T-DNA, respectively), which may be used as platforms for fluorescent codes. As a proof of concept, we have demonstrated seven different fluorescent codes with only two different fluorescent dyes using T-DNA. This method provides maximum efficiency for current flow cytometry. We are confident that this system will provide highly efficient multiplexed fluorescent detection for bioanalysis compared with one-to-one fluorescent correspondence for specific marker detection.     

A flow-cytometric method for determination of yeast viability and cell number in a brewery

A flow-cytometric assay, using the fluorescent dye, oxonol, for the simultaneous determination of yeast cell viability and cell number is described. The assay was optimised, and trialed at a brewery for 6 months. The flow-cytometry assay offered a substantially reduced error in viability determination, compared to methylene blue which is the industry standard for measuring viability. Further, by calculating yeast cell number at the same time, this assay provides a reliable method for determining pitching rate, allowing increased quality control of subsequent fermentations.