Quantification of metabolic activity of cultured plant cells by vital staining with fluorescein diacetate

The metabolic activity of suspension cultures of Sonneratia alba cells was quantified by measurement of the hydrolysis of fluorescein diacetate (FDA). FDA is incorporated into live cells and is converted into fluorescein by cellular hydrolysis. Aliquots (0.1–0.75 g) of S. alba cells were incubated with FDA at a final concentration of 222 μg/ml suspension for 60 min. Hydrolysis was stopped, and fluorescein was extracted by the addition of acetone and quantified by measurement of absorbance at 490 nm. Fluorescein was produced linearly with time and cell weight. Cells of S. alba are halophilic and proliferated well in medium containing 50 and 100 mM NaCl. Cells grown in medium containing 100 mM NaCl showed 2- to 3-fold higher FDA hydrolysis activity than those grown in NaCl-free medium. When S. alba cells grown in medium supplemented with 50 mM NaCl were transferred to fresh medium containing 100 mM mannitol, cellular FDA hydrolysis activity was down-regulated after 4 days of culture, indicating that the moderately halophilic S. alba cells were sensitive to osmotic stress. Quantification of cellular metabolic activity via the in vivo FDA hydrolysis assay provides a simple and rapid method for the determination of cellular activity under differing culture conditions.     

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     

Flow sorting and culture of protoplasts: Conditions for high-frequency recovery,growth and morphogenesis from sorted protoplasts of suspension cultures of nicotiana

Protoplasts were prepared from suspension cultures of Nicotiana tabacum cv Wisconsin 38 that had been prelabeled with FITC. The protoplasts were subjected to flow sorting based on fluorescence content using a Coulter EPICS V Flow Cytometer — Cell Sorter. Conditions were established that allowed the recovery after sorting of approximately 30% of the initial protoplasts in a viable state. These were subsequently regenerated into calli that underwent shoot morphogenesis.Abbreviations FITC Fluorescein isothiocyanate - FDA fluorescein diacetate     

Direct observations on generative cells isolated from pollen grains of Haemanthus katherinae baker

Generative cells from mature pollen grains of Haemanthus katherinae Baker (African blood lily) were isolated by means of a simple squash method and observed by differential interference contrast (DIC), fluorescence and polarizing microscopy. The isolated cells appeared structurally similar to those observed in vivo and gave no evidence of a typical cell wall. Their viability was confirmed using the fluorescein diacetate test. The cell shape changed rapidly as the sucrose concentration of the medium was varied. The squash method of isolating generative cells holds promise for the direct and experimental study of these cells, especially in the living state.Abbreviations FDA Fluorescein diacetate - PAS Periodic-acid-Schiff - DIC differential interference contrast - NA numerical aperture     

Plant regeneration from stem cortex protoplasts of a tomato hybrid

Longitudinal sections containing cortical cells taken from stem internodes of a hybrid betweenLycopersicon esculentum andSolanum lycopersicoides were used as tissue sources for enzymatic protoplast isolation. Greenhouse and growth room-grown plants 4–8 weeks after rooting could be used as sources of donor tissue. Protoplasts from these tissues divided within 2–4 days of culture and numerous microcalli formed within 30 days. The shoot regeneration frequency of protoplast-derived calli was in the order of 60%. More than 100 regenerated plants which appear phenotypically normal have been established in soil.Abbreviations NAA 1 naphthaleneacetic acid - 2,4-D 2-4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BA 6 benzylaminopurine - FDA fluorescein diacetate     

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.     

A rapid cell membrane permeability test using flourescent dyes and flow cytometry

A reliable and rapid test to detect cytotoxic chemicals which affect cell membranes is described. Fluorescein diacetate freely penetrates intact cells where it is hydrolyzed to its fluorochrome, fluorescein, which is retained in the cell due to its polarity. On the other hand, ethidium bromide is known to be excluded from the intact cell, staining only nucleic acids of membrane-damaged cells. The combination of both fluorochromes results in counter-staining: intact cells fluoresce green (cytoplasm) and membrane-damaged cells fluoresce red (nucleus and RNA). Rat thymocytes freshly isolated without enzyme treatment were incubated simultaneously with test substance and dye solution fluorescein diacetate and ethidium bromide. A two-parameter analysis was performed on a flow cytometer with an on-line computer. Concentration-dependent effects of various detergents and solvents were quantified by measuring the amount of dye retention, i.e., the decrease or increase in fluorescein—fluorescence (peak shift), and the decrease in dye exclusion (increase in ethidium bromide-staining) relative to the untreated control. The assay can be used for rapid monitoring of chemical insults to cell membranes which precede the decrease of the viability measured by pure dye exclusion techniques.Abbreviations DMA dimethyl sulfate - DMSO dimethyl sulfoxide - EB ethidium bromide - F fluorescein - FDA fluorescein diacetate - FS₂₅ concentration of test substance resulting in a F-peak left-shift of 25% from control - PBS phosphate buffered saline - SCT forward light scatter - SDS sodium dodecyl sulfate     

Plant regeneration from stem cortex expiant and protoplast cultures of Brassica juncea (mustard)

Summary Plant regeneration from stem cortex explants of 13 genotypes of Brassica juncea was assessed. Regeneration was strongly affected by genotype, as up to 50.6 shoots were produced per 100 calli of the most responsive line (Blaze), whereas no shoots were obtained from less responsive lines (Zeml, Vniimk351). Blaze was chosen for B. juncea stem cortex protoplast isolation. After one week of culture, 11–14% of the cells had divided, and about 0.002% produced 1–2 mm colonies within 6 weeks. Up to 7% of these colonies gave rise to shoots upon transfer to plant regeneration medium.Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - BAP 6-benzylaminopurine - FDA fluorescein diacetate - IAA indole acetic acid - IBA indole butyric acid - 2ip 2-isopentenyl-adenine - NAA 1-naphthaleneacetic acid - TD thidiazuron     

Isolation and cell regeneration of protoplasts from sugar pine (Pinus lambertiana)

Protoplasts were isolated at high yields from actively growing callus and cell suspensions of cotyledons and needles of mature trees. The best protoplast growth response was obtained from cell suspensions of cotyledon and needle callus. Lower protoplast yields were obtained directly from young needles of flushing buds on explants from mature shoots (30-year-old trees) growing in vitro. In all cases, the first divisions, promoted by dimethyl sulfoxide, were observed in 10–45% of the protoplasts by 7–10 days. After 25–30 days, colonies of 8–10 cells were established. Browning of protoplast-derived cell cultures was observed within 40–45 days (cotyledons) and 20–25 days (mature tree sources).Abbreviations BA N⁶-Benzyladenine - DCR Douglas-fir cotyledon revised medium - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - FDA Fluorescein diacetate - Mes 2-(N-morpholino) ethanesulfonic acid - NAA -naphthaleneacetic acid     

Analysis of enzyme kinetics in individual living cells utilizing fluorescence intensity and polarization measurements

BACKGROUND: The Cellscan mark-S (CS-S) scanning cytometer was used for tracing enzymatic reactions in the same individual cells under various physiological conditions over periods of minutes. On-line reagent addition and changes in the experimental conditions (buffers, ions, substrates and inhibitors) were performed. METHODS: Kinetic events were monitored by fluorescence intensity (FI) and fluorescence polarization (FP) measurements of fluorescein diacetate (FDA) and chloromethyl fluorescein diacetate (CMFDA) intracellular hydrolysis. FP measurements have been used to assess the intracellular marker's mobility restrictions. RESULTS: Kinetic measurement along 1000 s of FDA labeled individual Jurkat T cells, indicated variation of 65% for FI(t) and approximately 10% for FP(t). While FI increased linearly with time, FP(t) decreased nonlinearly and asymptotically, reaching a constant value. The FP(t) of CMFDA-labeled cells was different from that of FDA-labeled cells. Average cellular Km of 3.9 microM was calculated from individual cell FDA hydrolysis curves. CONCLUSIONS: (1) Analysis of the reaction kinetics of intracellular enzymes can be refined by using FP measurements of the products of fluorogenic substrates in addition to the FI measurements. (2) Subpopulations or individual cells could be classified according to their reaction rates. (3) A specific dependence of FP(t) on type of enzyme substrate is suggested.     

Microbial cell responses to a non-ionic surfactant. II. Effects as assessed by fluorescein diacetate uptake

Summary The effects of a non-ionic surfactant, Pluronic F-68, on uptake of fluorescein diacetate (FDA) into yeast cells as measured by increase in fluorescence atca. 500 nm have been studied. The rate of FDA uptake was increased almost 2.5 fold by incubating cells with up to 5% commercial grade pluronic but this depended on source and degree of purity. Dye uptake was reduced by pre-purification of pluronic but this again depended on source of material. None of the pluronic preparations had any significant effects on the rate of enzymemediated FDA hydrolysis by cell-free extracts.     

Fluorometric estimation of viable animal cells immobilized in alginate gel

Fluorescein diacetate (FDA) is hydrolyzed to a fluorescent compound, fluorescein (FRC) which is retained in viable animal cells having an intact plasma membrane. The FDA-FRC system was applied to analysis of viable cells for a mouse hybridoma 16-3F cell entrapped in alginate gel. As a result, visualization of the viable cell distribution in the gel matrix became possible. Moreover, a linear relationship was confirmed between the viable cell concentration determined by microscopic counting and the fluorescence intensity derived from FRC in the viable cells. Thus, cell growth could be quantitatively estimated on the basis of the fluorometry for the immobilized-cell culture.     

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.     

Fluorescein Diacetate Hydrolysis as a Measure of Total Microbial Activity in Soil and Litter

Spectrophotometric determination of the hydrolysis of fluorescein diacetate (FDA) was shown to be a simple, sensitive, and rapid method for determining microbial activity in soil and litter. FDA hydrolysis was studied in soil and straw incubated for up to 3 h. Hydrolysis was found to increase linearly with soil addition. FDA hydrolysis by pure cultures of Fusarium culmorum increased linearly with mycelium addition both in shake cultures and after inoculation into sterile soil. FDA hydrolysis by Pseudomonas denitrificans increased linearly with biomass addition. The FDA hydrolytic activities in soil samples from different layers of an agricultural soil were correlated with respiration. Acetone was found to be suitable for terminating the reaction.     

Optimisation of the fluorescein diacetate antibacterial assay

The fluorescein diacetate (FDA) antibacterial assay relies on the cleavage of fluorescein diacetate by metabolically active bacteria. The recent finding that microbiological media can lead to significant levels of cleavage has reduced the reliability of the assay. Using the nucleophilic scavengers N-ethylmaleimide and maleic anhydride, we have demonstrated that this abiotic cleavage is most likely due to nucleophiles such as cysteine and histidine commonly present in the media. To increase the reliability of the assay we have modified the original assay conditions to include use of dilute medium (peptone 0.2% w/v, yeast extract 0.1% w/v and NaCl 0.1% w/v) in a non-nucleophilic buffer and overnight incubation of the medium after addition of antibacterial agents. The optimised fluorescein diacetate assay has been used to determine the MIC of gentamicin, tetracycline and chloramphenicol for Escherichia coli, Staphyloccocus aureus and Pseudomonas aeruginosa and gave quantitative results that were reproducible and consistent with published data.     

Colonization,biofilm formation and biodegradation of polyethylene by a strain of<Emphasis Type="Italic"> Rhodococcus ruber</Emphasis>

A two-step enrichment procedure led to the isolation of a strain of Rhodococcus ruber (C208) that utilized polyethylene films as sole carbon source. In liquid culture, C208 formed a biofilm on the polyethylene surface and degraded up to 8% (gravimetrically) of the polyolefin within 30 days of incubation. The bacterial adhesion to hydrocarbon assay and the salt aggregation test both showed that the cell-surface hydrophobicity of C208 was higher than that of three other isolates which were obtained from the same consortium but were less efficient than C208 in the degradation of polyethylene. Mineral oil, but not nonionic surfactants, enhanced the colonization of polyethylene and increased biodegradation by about 50%. Fluorescein diacetate (FDA) hydrolysis and protein content analysis were used to test the viability and biomass density of the C208 biofilm on the polyethylene, respectively. Both FDA activity and protein content of the biofilm in a medium containing mineral oil peaked 48–72 h after inoculation and then decreased sharply. This finding apparently reflected rapid utilization of the mineral oil adhering to the polyethylene. The remaining biofilm population continued to proliferate moderately and presumably played a major role in biodegradation of the polyethylene. Fourier transform infrared spectra of UV-photooxidized polyethylene incubated with C208 indicated that biodegradation was initiated by utilization of the carbonyl residues formed in the photooxidized polyethylene     

A comparative kinetic study on the conversion of fluoresceindiacetate to fluorescein in living cells and in vitro

Summary The kinetics of fluoresceindiacetate (FDA)-hydrolysis in erythrocytes, CHO-cells and homogenized cells was investigated by means of fluorescence spectroscopy. At a 10^–6 m FDA concentration the enzymatic conversion in the homogenized cells can be described by first order reactions. When studying intact cells, total kinetics requires the addition of a first order transport term. Consequently, the total hydrolytic process can be described by three exponentials. The following model was set up on the basis of the data obtained: FDA, which exists in two sterically differing conformations, reaches the cell via an active transport mechanism, where the two conformations are converted at different rates into fluorescein by an enzymatic reaction. Intracellular FDA-enrichment factors of 100–600 were derived. In addition, the action of chemical inhibitors and of elevated temperatures on the individual reaction steps was investigated.     

Sugarcane protoplasts: factors affecting division and plant regeneration

Summary Sugarcane cell suspensions were initiated from leaf callus and sub-cultured every 7 to 10 days by alternate transfer to MS based medium with 3.0 and 1.0 mg 1^–12,4-D. Suspensions older than 3 months gave the most reproducible yields of protoplasts. Isolated protoplasts required 50 mM Ca²⁺ in the washing solution and 100 mM Ca²⁺ in the culture medium to prevent lysis. At plating densities of 2.0–3.0×10⁵ ml^–1, 18% or more of the isolated protoplasts produced cell colonies when cultured in droplets or sectors of Kao and Michayluk (1975) based medium with 1.2% w/v Sea Plaque agarose. Cell colonies were of two morphological types. Those consisting of small, tightly packed cells developed into morphogenic callus. The latter produced an abundance of green meristems from which shoots and whole plants were regenerated.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - MS Murashige and Skoog (1962)     

Protoplast production in Chondrus crispus gametophytes (gigartinales,rhodophyta)

Protoplasts were isolated from female gametophytes of Chondrus crispus (Stackh.) using commercial cellulase and various carrageenases prepared from marine bacteria. Depending on the nature of the donor tissue (apices or whole thallus, wild or cultivated strains), yields ranged from 1.0–8.5×10⁸ protoplasts per gram of fresh tissue. Preincubating the tissue with a potassium chelator, Kryptofix 222, enhanced protoplast yields by 30–50 %. Based on staining with fluorescein diacetate most protoplasts were viable. A few protoplasts regenerated a cell wall and divided.     

Loading process of sugars into cabbage petiole and asparagus shoot apex cells by incubation with hypertonic sugar solutions

Summary The freezing tolerance of cabbage petioles and asparagus shoot apexes was increased by preincubation with 0.8 M sugar solutions. In cabbage petioles with an initial freezing tolerance of –3 °C (temperature for 50% cell survival), as determined by both electrolyte leakage and fluorescein diacetate vital staining, the freezing tolerance was increased to –13 °C by incubation with sorbitol solutions for 3 h. In meristematic cells of asparagus shoot apexes with an initial freezing tolerance of –7.5 °C, as determined by fluorescein diacetate vital staining, the freezing tolerance was increased to –30 °C by incubation with 0.8 M sugar solutions for 3 h, although other cells in the shoot apexes were killed by higher freezing temperatures. During incubation of both cabbage petioles and asparagus shoot apexes with sugar solutions, sugars were intracellularly taken up by osmotically induced fluid-phase endocytotic vesicles, as indicated by comovement of Lucifer Yellows carbohydrazide (LYCH) observed with a confocal laser scanning microscope. The amounts of intracellularly taken up sugars increased concomitantly with the formation of endocytotic vesicles depending on the time of incubation in parallel with a gradual increase of freezing tolerance. However, the endocytotic vesicles and their contents were retained not only after prolonged incubation after maximum freezing tolerance had been achieved but also after recovery of these tissue cells to isotonic conditions or after freeze-thawing. These results suggest that although sugars are intracellularly taken up by endocytotic vesicles, they might be sequestered within vesicles, casting doubt on their protective role to the plasma membranes as a main site of freezing injury. The pretreatment with 1 mMp-chloromercuribenzenesulfonic acid (PCMBS), an inhibitor of sugar transport, reduced the amounts of intracellular sugar uptake without affecting the formation of endocytotic vesicles, suggesting that sugars were, at least partly, taken up by sugar transporters. In the pretreatment with PCMBS, the freezing tolerance of incubated tissues with sugar solutions was significantly reduced, although addition of PCMBS per se did not affect survival. These results suggest that sugars taken up by sugar transporters, rather than sugars taken up by endocytotic vesicles, are mainly responsible for the increased freezing tolerance of cabbage petioles and asparagus shoot apexes. Furthermore, we aimed to study the occurrence of fluid-phase endocytosis with LYCH in an isotonic condition. Our results indicated that uptake of LYCH by fluid-phase endocytotic vesicles was not detected microscopically in isotonic condition, although LYCH was spectrofluorimetrically taken up in isotonic condition. Spectrofluorimetric uptake of LYCH was inhibited by addition of probenecid, an anion transport inhibitor. These results suggest that in cabbage petioles and asparagus shoot apexes, LYCH is taken up by anion transport but not by fluid-phase endocytosis in isotonic condition, and uptake of LYCH by fluid-phase endocytosis is restricted to occur only in hypertonic condition.Abbreviations CLSM confocal laser scanning microscope - FDA fluorescein diacetate - LYCH Lucifer Yellow carbohydrazide - PCMSB p-chloromercuribenzenesulfonic acid - T_EL50 temperature at which 50% electrolyte leakage occurred