Fluorimetric quantification of cell death in monolayer cultures and cell suspensions.

A fluorimetric assay using ethidium bromide (EB) was employed to quantify cell death in monolayer cell cultures (MA-104 cells) in situ and isolated cell suspensions (isolated colonic cells and Leishmania). Fluorescence of EB stained cells was measured with a photometer coupled to an inverted microscope for cell monolayers or in a spectrofluorometer for cell suspensions. Dead cells stained with trypan blue were fluorescent with EB in all preparations studied, but the latter gave an unequivocal signal. Staining with EB and fluorescein diacetate was mutually exclusive. The relationship between the number of EB fluorescent cells and the intensity of fluorescence measured in the microphotometer was linear for a large range of cell numbers (1-14000) from different types of preparations. Applicability of the method for measuring living and dead cells in two different time scales (minutes and hours) is shown using MA-104 cell monolayers infected with rotavirus and Leishmania suspensions treated with amphotericin B. The method is fast, simple, sensitive and reliable, enabling quantification of living and dead cells in monolayers and suspensions.     

Fluorescein diacetate used as a vital stain for labeling living pollen tubes

Snapdragon and tobacco pollen treated with fluorescein diacetate (FDA, 10 μg/ml) for 40 min could germinate and grew well in FDA-free medium. The pollen tubes showed bright fluorescence of the protoplasm when they were living. Thus the FDA method can be used, in addition to its previous usage for viability test of cells, also for vital staining of pollen tubes, and may be valuable to the study of other living cells as well.     

Potential problems with fluorescein diacetate assays of cell viability when testing natural products for antimicrobial activity.

There are two potential problems in the use of fluorescein diacetate (FDA) as a measure of cell viability. The first is the hydrolysis of FDA to fluorescein in the absence of live cells and the second is the quenching of fluorescence by assay solutions. We show that common media components such as tryptone, peptone and yeast extract all promote hydrolysis of FDA in the absence of live cells, as do Tris-HCl and sodium phosphate buffers. As a consequence, various microbiological media promote hydrolysis of FDA in the absence of live cells. Different media were also shown to reduce the amount of visible fluorescence of fluorescein. Diluting the medium decreases the background hydrolysis of FDA as well as increases the amount of visible fluorescence. Both problems should be considered when using FDA as an indicator of cell viability when testing natural products for antimicrobial activity.     

Elicitation of the Hypersensitive Responses in Tabacco by a10.6 kD Proteinaceous Elicitor from Phytophthora palmi

A 10.6 kD heat resistant, proteinacious elicitor was purified from the culture filtrate of Phytophthora palmivora Butler but not from P. melonis Katsura. The 10.6 kD elicitor is a holoprotein devoid of glycoside. It can cause hypersensitive necrosis of the detached tobacco ( Nicotiatna tabacum L. ) leaves 48 h post-inoculation with dosages of above 40 μg. Four cell types were investigated by using Confocal microscopy, and 2' ,7'-dichlorodihydrofluorescein diacetate (DCFDA) as a probe of H2O2 production. It was showed that oxidative burst occurred in cultured suspension cells as well as in mesophyll cells, epidermal cells and guard cells within 10 min upon the elicitor treatment. The hypersensitive cell death appeared 6 h after the treatment when inoculated with fluorescein diacetate (FDA) as indicator of cell viability. These results suggest that H2O2 accumulation was the main cause of the hypersensitive cell death in tobacco induced by the 10.6 kD elicitor. This 10.6 kD elicitor may belong to the family of elicitins.     

应用流式细胞术检测毕赤酵母的细胞活性

选取两种细胞活性染色试剂二乙酸荧光素(fluoresceindiacetate,FDA)和碘化丙锭(propidiumiodide,PI),应用流式细胞术(flowcytometry,FCM)检测毕赤酵母细胞活性。比较FDA/PI双染色与PI单染色的FCM图谱,后者能够很好地将死活细胞区分开来并得到正确的比例。利用PI单染色检测发酵过程细胞活性的变化,甘油补料阶段几乎没有细胞死亡,进入甲醇补料阶段后,随着细胞密度的增加,细胞的活性不断降低,发酵88h时细胞活性仅为73.8%。     

Phytophthora palmi分泌的10.6kD蛋白激发烟草的过敏反应

从疫霉菌Ｐｈｙｔｏｐｈｔｈｏｒａ ｐａｌｍｉｖｏｒａ Ｂｕｔｌｅｒ的培养滤液中分离出分子量为１０．６ｋＤ的不含糖基的耐热蛋白．这种１０．６ｋ蛋白能诱导烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）叶片发生过敏性坏死反应。而疫霉菌另一种Ｐ．ｍｅｌｏｎｉｓ Ｋａｔｓｕｒａ的培养滤液中不含这种类似蛋白,不能诱导烟草叶片发生过敏反应。利用共聚焦激光扫描显微镜,以荧光探剂ＦＤＡ（ｆｌｕｏｒｅｓｃｅｉｎ ｄ     

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.     

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.     

Staining with Fluorescein Diacetate Correlates with Hepatocyte Function

To establish the importance of fluorescein diacetate (FDA) as a viability stain for cultured hepatocytes. we hypothesized that FDA staining would correlate positively with hepatocyte viability and function. Mixtures of live and dead cells were stained with FDA and scanned by flow cytometry. A close correlation was observed between the live cell fraction and percent viability as determined by FDA staining (R² = 0.962). Hepatocytes were also sorted into low fluorescence and high fluorescence groups. Both albumin production and lidocaine metabolism (P-450 activity) were significantly increased in the high fluorescence group compared to the low fluorescence group. An automated, fluorescence-activated assay was useful for rapid assessment of hepatocyte viability. In addition. the intensity of green fluorescence following staining with FDA correlated well with two specific measures of hepatocyte function.     

The life and death of sponge cells

Cell viability is an essential touchstone in the study of the effect of medium components on cell physiology. We developed a flow-cytometric assay to determine sponge-cell viability, based on the combined use of fluorescein diacetate (FDA) and propidium iodide (PI). Cell fluorescence measurements based on incubation of cells with FDA or PI resulted in a useful and reproducible estimate of the viability of primary sponge-cell cultures. We studied the effects of temperature, ammonium, and the fungicide amphotericin B on the viability of a primary-cell culture from the marine sponge Suberites domuncula using the aforementioned flow-cytometric assay. S. domuncula cells die rapidly at a temperature of >or=22 degrees C, but they are insensitive to ammonium concentrations of up to 25 mM. Amphotericin B, which is frequently used in sponge-cell culture media, was found to be toxic to S. domuncula cells.     

A Method for Producing Dust-, Streak- and Hole-Free Formvar Films in Laboratories Having High Atmospheric Humidity

To establish the importance of fluorescein diacetate (FDA) as a viability stain for cultured hepatocytes. we hypothesized that FDA staining would correlate positively with hepatocyte viability and function. Mixtures of live and dead cells were stained with FDA and scanned by flow cytometry. A close correlation was observed between the live cell fraction and percent viability as determined by FDA staining (R² = 0.962). Hepatocytes were also sorted into low fluorescence and high fluorescence groups. Both albumin production and lidocaine metabolism (P-450 activity) were significantly increased in the high fluorescence group compared to the low fluorescence group. An automated, fluorescence-activated assay was useful for rapid assessment of hepatocyte viability. In addition. the intensity of green fluorescence following staining with FDA correlated well with two specific measures of hepatocyte function.     

Isolation and purification of generative cells from fresh pollen of Vicia faba L.

A technique named two-step osmotic shock was developed for isolating generative cells (GCs): pollen grains were incubated in 20% sucrose solution and shocked by adding water. This caused the pollen grains to burst and release their contents including GCs. By subsequent filtration and centrifugation the isolated GCs were purified. The viability of GCs before and after purification was confirmed by fluorescein diacetate (FDA) test. This procedure offers a rapid and effective means to obtain living GCs in quantities.Abbreviations GC Generative cell - FDA Fluorescein diacetate     

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.     

Quantification of an in vitro cell-cell adhesion assay using interactive laser scanning cytometry.

We are interested in identifying cell-cell adhesion molecules on the surface of Sertoli cells that mediate Sertoli cell-spermatogenic cell adhesion. Numerous cell-cell adhesion assays employ microscopic observation, photomicroscopy or radioactive isotopes for quantification. Previously, we developed an in vitro assay for testicular cell interactions. This assay was, however, time consuming using photography for analysis. We have now modified this system using laser cytometry to quantify adherent cells. Rat testicular epithelial cells are cultured for approximately 6 days before labelling with fluorescein diactetate (FDA) to assess confluency by image scanning so that spermatogenic cell binding can be normalized to available epithelial cell surface area. Rat spermatogenic cells are labeled with FDA before addition to epithelial cell monolayers. In some studies, purified spermatogenic cell populations were isolated to determine average cell size. We found that spermatocyte area varied between 225-500 microns2, spermatids were 100-225 microns2 and residual bodies were less than 100 microns2. Using these parameters, scanning cytometry allows the differential analysis of adhesion by individual germ cell sub-classes from mixed cell suspensions, saving time, animals, and major expense. The scanning laser assisted assay is faster, more reproducible and less subjective than earlier cell-cell adhesion assays using light microscopy or isotopes. This experimental approach should facilitate any cell-cell adhesion assay in which one cell type is adherent to a substrate.     

Rapid Determination of Conidial Viability for Entomopathogenic Hyphomycetes Using Fluorescence Microscopy Techniques

The viability of conidia from two species of deuteromycetes fungi pathogenic to insects was determined using two fluorochrome stains, fluorescein diacetate (FDA) and propidium iodide (PI). These stains were used either alone or in combination, and results were compared with standard conidial germination tests. FDA fluoresces bright green in viable conidia and PI fluoresces red in non-viable conidia, when viewed using specific fluorescence microscopic techniques. Conidia from two isolates of Paecilomyces fumosoroseus (Wize) Brown and Smith and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated. Conidia were suspended in deionized water and half of each suspension was treated with microwave radiation to kill all the conidia. Conidia were tested for viability in non-microwaved suspensions in a mixture (ca. 1:1) of viable and non-viable conidial suspensions, and in the microwaved suspensions that contained all non-viable conidia. No significant differences were observed for the four isolates tested between germination tests on water and agar and viability tests conducted with FDA alone or FDA in combination with PI. One isolate of B. bassiana that had been damaged in storage was also tested. Differences were observed between the actual germination and the percentage of viability determined using FDA or FDA plus PI. Damaged conidia maintained a measure of viability and fluoresced green, but did not fully germinate.     

The Use of Fluorescein Diacetate and Ethidium Bromide as a Viability Stain for Isolated Islets of Langerhans

There is a need for a simple, rapid, sensitive method for assessing the viability of isolated islets of Langerhans. In this study the fluorescent dyes fluorescein diacetate (FDA) and ethidium bromide (EB) have been used to provide a viability assay for isolated rat islets. Discrimination of living from dead islets is efficient; in a blind sorting experiment using freshly isolated islets and islets killed by either heat or alcohol, viability determined by insulin secretion in response to glucose stimulation correlated well with viability as determined by FDA/EB staining. Furthermore, it is possible to discriminate degrees of viability, and a scoring system is described for this purpose which is shown to correlate with another index of viability, the ATF content A reliable viability stain should not itself be toxic; FDA/EB stained islets remain viable after staining, showing normal response to glucose stimulation and normal function after transplantation.     

Single-cell viability assessment with a novel spectro-imaging system

Single-cell viability assessment by means of plural dye probes require the spectral and temporal analysis of microscopic images of the test cells. To meet this requirement, we have developed a simple and compact spectro-imaging system using an image slicer and a grism. The image slicer was made of a bundle of 100 optical fibers. The field of view is divided into 10 x 10 sections. The spectral data of each section could be recorded every 5 s in the range from 400 to 800 nm at 5 nm resolution. The viability changes of yeast or tobacco single-cells were measured with this system. Using BY-2 cells, for example, the response to a chemical stress of saponin was measured by means of two fluorescent probes. The spectral-spatial-temporal data of fluorescein and DNA bound ethidium bromide provided us with useful information about the dynamic change of cell membrane permeability from which the cell viability was assessed.     

Cadmium toxicity in cultured tomato cells--role of ethylene, proteases and oxidative stress in cell death signaling

Our aim was to investigate the ability of cadmium to induce programmed cell death in tomato suspension cells and to determine the involvement of proteolysis, oxidative stress and ethylene. Tomato suspension cells were exposed to treatments with CdSO(4) and cell death was calculated after fluorescein diacetate staining of the living cells. Ethylene was measured in a flow-through system using a laser-driven photo acoustic detector; hydrogen peroxide was determined by chemiluminescence in a ferricyanide-catalysed oxidation of luminol. We have demonstrated that cadmium induces cell death in tomato suspension cells involving caspase-like proteases, indicating that programmed cell death took place. Using range of inhibitors, we found that cysteine and serine peptidases, oxidative stress, calcium and ethylene are players in the cadmium-induced cell death signaling. Cadmium-induced cell death in tomato suspension cells exhibits morphological and biochemical similarities to plant hypersensitive response and to cadmium effects in animal systems.     

High-throughput screening of cell death inducible short peptides from TNF-related apoptosis-inducing ligand sequence

Therapeutic peptides and small molecules, rationally designed to trigger cell death have attracted strong attention. Cell death inducible peptides were screened from amino acid sequence of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Using Fmoc solid phase synthesis, cellulose membrane-bound octameric peptide library of TRAIL scan was prepared and cell viability assay was directly performed on peptide disk with Jurkat cells. Six peptide sequences that could induce cell death were found. Peptide sequence with RNSCWSKD (TRAIL(227-234)) that exist in the zinc-binding site revealed high cell death inducible activity. Apoptotic cell death was observed when cells were treated with soluble synthesized peptide.     

The use of fluorescein diacetate and ethidium bromide as a viability stain for isolated islets of Langerhans

There is a need for a simple, rapid, sensitive method for assessing the viability of isolated islets of Langerhans. In this study the fluorescent dyes fluorescein diacetate (FDA) and ethidium bromide (EB) have been used to provide a viability assay for isolated rat islets. Discrimination of living from dead islets is efficient; in a blind sorting experiment using freshly isolated islets and islets killed by either heat or alcohol, viability determined by insulin secretion in response to glucose stimulation correlated well with viability as determined by FDA/EB staining. Furthermore, it is possible to discriminate degrees of viability, and a scoring system is described for this purpose which is shown to correlate with another index of viability, the ATP content. A reliable viability stain should not itself be toxic; FDA/EB stained islets remain viable after staining, showing normal response to glucose stimulation and normal function after transplantation.