Vital Fluorochrome Research Articles

Optimal fixation period of a combined bioconstruction with buccal epithelial cells in limbal cell deficiency in experiment

BACKGROUND: The search for an effective method for the treatment of limbal stem cell deficiency of various etiologies, leading to intense clouding and vascularization of the cornea, followed by a significant decrease in visual acuity, remains an important and relevant topic in ophthalmology. The results of the studies showed that transplantation of buccal epithelial cells could significantly improve the prognosis of treatment in this category of patients.
 AIM: To determine the optimal fixation period of a combined bioconstruction with buccal epithelial cells for the treatment of limbal cell deficiency in experiment.
 MATERIALS AND METHODS: At the first stage, the corneal epithelium was removed with a scraper in the eyes of experimental animals (12 eyes), and the limb was excised along the entire circumference. Next, to isolate epithelial buccal cells and manufacture a combined bioconstruction consisting of buccal cells, a collagen carrier and a soft contact lens, a flap of the mucous membrane of 5 5 mm was taken from the cheeks of rabbits. At the second stage, after the formation of a fibrovascular pannus on the cornea, it was excised to transparent layers of the cornea, a combined bioconstruction was placed on top. Further, one U-shaped suture was applied at the border of the inner and outer third of the eyelids. Temporary blepharorraphia persisted for 3 days (6 eyes) and 5 days (6 eyes), after the specified time, stitches were removed from the eyelids, and bioconstructions were removed. The bioconstructions removed from the eyes were stained with a vital (lifetime) fluorochrome dye based on tripaflavin and acridine orange, followed by analysis in a fluorescent microscope. The structure of cell nuclei, the overall integrity of the cytoplasm, and the presence of secretory vesicles were evaluated. After 7 and 14 days after transplantation of the bioconstruction, the area of erosion, the extent of new vessels and the transparency of the cornea were evaluated. When the observation was completed, the rabbits were removed from the experiment, and the eyes were enucleated and subjected to histological examination.
 RESULTS: On the surface of all bioconstructions removed after 5 days, only leukocytes were detected. At the same time, on collagen matrix of bioconstructions removed after 3 days, in addition to leukocytes, there were also buccal epithelial cells with normal nuclear structure and chromatin topography in its composition, as well as with secretory vesicles. On days 714, both groups showed a decrease in the area of erosion, while the dynamics of ocular surface recovery processes was more pronounced in the group with fixation of the bioconstruction for 5 days.
 CONCLUSIONS: According to the results of in vivo staining with fluorochrome dye of combined bioconstructions removed from the cornea of experimental animals 3 and 5 days after superficial keratectomy, as well as the data of clinical and histological examination, the optimal period for bioconstruction fixation was established to be 5 days.

Use of live, fluorescently-labeled algae for measuring microzooplankton grazing in natural communities

Here, our goal was to develop a technique for staining live algae with vital fluorochromes and to further test whether this method may serve as a tool for examining the trophic roles and functional diversity of microzooplankton. We tested 4 fluorochromes on a total of 10 phytoplankton species, out of which only 3 proved effective, and only on some of the species tested. The fluorochrome Vybrant did not dye any algal species, CellTracker Blue successfully dyed 2 nanoflagellate species (Isochrysis galbana and Tetraselmis sp.) and one dinoflagellate (Heterocapsa sp.), and LysoSensor and LysoTracker each dyed 2 diatom species (Thalassiosira weissflogii and Skeletonema costatum). Further experiments with the 2 most successful fluorochromes (CellTracker and LysoSensor) indicated that optimum incubation times ranged from 4 to 8h and that the percentage of stained cells was not improved at concentrations higher than 10μM and 2μM for CellTracker and LysoSensor, respectively. The residence times of the fluorochromes under natural light conditions were greater than 24h (60–80% of stained algae). Labeling algae with CellTracker had no significant effect on their growth rate or C:N molar ratio. LysoSensor, however, had minor (although significant) effects on the growth rates of stained vs. unstained algae. Bottle grazing experiments showed that Oxyrrhis marina grazed on unstained nanoflagellate species at equal rates to those stained with CellTracker; however, a positive discrimination for stained cells was detected when Gyrodinium dominans was used as the grazer. We also measured microzooplankton ingestion rates in natural algal communities by combining the dilution method with the addition of live algae into a natural plankton suspension. The addition of stained algae did not significantly affect phytoplankton growth or mortality rates due to microzooplankton grazing. The low toxicity of fluorochromes and the easy visualization of labeled algae inside predators make this method a useful tool for estimating grazing rates of microzooplankton and for the quantification of different trophic interactions among protists in the microbial food web. However, given the limited number of algae species successfully stained further research is needed to obtain more universal dyes.

Quantitative Assessment of Mycoplasma Hemadsorption Activity by Flow Cytometry

A number of adherent mycoplasmas have developed highly complex polar structures that are involved in diverse aspects of the biology of these microorganisms and play a key role as virulence factors by promoting adhesion to host cells in the first stages of infection. Attachment activity of mycoplasma cells has been traditionally investigated by determining their hemadsorption ability to red blood cells and it is a distinctive trait widely examined when characterizing the different mycoplasma species. Despite the fact that protocols to qualitatively determine the hemadsorption or hemagglutination of mycoplasmas are straightforward, current methods when investigating hemadsorption at the quantitative level are expensive and poorly reproducible. By using flow cytometry, we have developed a procedure to quantify rapidly and accurately the hemadsorption activity of mycoplasmas in the presence of SYBR Green I, a vital fluorochrome that stains nucleic acids, allowing to resolve erythrocyte and mycoplasma cells by their different size and fluorescence. This method is very reproducible and permits the kinetic analysis of the obtained data and a precise hemadsorption quantification based on standard binding parameters such as the dissociation constant K d. The procedure we developed could be easily implemented in a standardized assay to test the hemadsorption activity of the growing number of clinical isolates and mutant strains of different mycoplasma species, providing valuable data about the virulence of these microorganisms.

Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye

Availability of purified or highly enriched fractions representing the various spermatogenic stages is a usual requirement to study mammalian spermatogenesis at the molecular level. Fast preparation of high quality testicular cell suspensions is crucial when flow cytometry (FCM) is chosen to accomplish the stage/s purification. Formerly, we reported a method to rapidly obtain good quality rodent testicular cell suspensions for FCM analysis and sorting. Using that method we could distinguish and purify early meiocytes (leptotene/zygotene stages, L/Z) from more advanced ones (pachytene, P) in guinea pig, which presents an unusually high content of early stages. Here we present an upgrade of that method with improvements that enabled the obtainment of high-purity meiotic substages also from mouse testis, namely:•Shortening of the mechanical disaggregation time to optimize the integrity of the suspension.•Elimination of the 25μm-filtration step to ensure the presence of large P cells.•Inclusion of a non-cytotoxic, DNA-specific, 488nm-excitable vital fluorochrome (Vybrant DyeCycle Green [VDG], Invitrogen) instead of Hoechst 33342 (requires UV laser, which can damage nucleic acids) or propidium iodide (usually related to dead/damaged cells). As far as we know, this is the first report on the use of this fluorochrome for the discrimination and purification of meiotic prophase I substages.

Fluorochrome-Functionalized Nanoparticles for Imaging DNA in Biological Systems

Attaching DNA binding fluorochromes to nanoparticles (NPs) provides a way of obtaining NPs that bind to DNA through fluorochrome mediated interactions. To obtain a nanoparticle (NP) that bound to the DNA in biological systems, we attached the DNA binding fluorochrome, TO-PRO 1 (TO), to the surface of the Feraheme (FH) NP, to obtain a fluorochrome-functionalized NP denoted TO-FH. When reacted with DNA in vitro, TO-FH formed microaggregates that were characterized by fluorescence, light scattering, and T2 changes. The formation of DNA/TO-FH microaggregates was also characterized by AFM, with microaggregates exhibiting a median size of 200 nm, and consisting of DNA and multiple TO-FH NPs whose individual diameters were only 25-35 nm. TO-FH failed to bind normal cells in culture, but treatment with chemotherapeutic agents or detergents yielded necrotic cells that bound TO-FH and vital fluorochromes similarly. The uptake of TO-FH by HT-29 xenografts (treated with 5-FU and oxaliplatin) was evident by surface fluorescence and MRI. Attaching multiple DNA binding fluorochromes to magnetic nanoparticles provides a way of generating DNA binding NPs that can be used to detect DNA detection by microaggregate formation in vitro, for imaging the DNA of necrotic cells in culture, and for imaging the DNA of a tumor treated with a chemotherapeutic agent. Fluorochrome functionalized NPs are a multimodal (magnetic and fluorescent), highly multivalent (n ≈ 10 fluorochromes/NP) nanomaterials useful for imaging the DNA of biological systems.

The slow cell death response when screening chemotherapeutic agents

To examine the correlation between cell death and a common surrogate of death used in screening assays, we compared cell death responses to those obtained with the sulforhodamine B (SRB) cell protein-based "cytotoxicity" assay. With the SRB assay, the Hill equation was used to obtain an IC50 and final cell mass, or cell mass present at infinite agent concentrations, with eight adherent cell lines and four agents (32 agent/cell combinations). Cells were treated with high agent concentrations (well above the SRB IC50) and the death response determined as the time-dependent decrease in cells failing to bind both annexin V and vital fluorochromes by flow cytometry. Death kinetics were categorized as fast (5/32) (similar to the reference nonadherent Jurkat line), slow (17/32), or none (10/32), despite positive responses in the SRB assay in all cases. With slow cell death, a single exposure to a chemotherapeutic agent caused a slow, progressive increase in dead (necrotic) and dying (apoptotic) cells for at least 72h. Cell death (defined by annexin and/or fluorochrome binding) did not correlate with the standard SRB "cytotoxicity" assay. With the slow cell death response, a single exposure to an agent caused a slow conversion from vital to apoptotic and necrotic cells over at least 72h (the longest time point examined). Here, increasing the time of exposure to agent concentrations modestly above the SRB IC50 provides a method of maximizing cell kill. If tumors respond similarly, sustained low doses of chemotherapeutic agents, rather than a log-kill, maximum tolerated dose strategy may be an optimal strategy of maximizing tumor cell death.

Re‐evaluating acridine orange for rapid flow cytometric enumeration of parasitemia in malaria‐infected rodents

Methods facilitating research in malaria are of pivotal relevance. Flow cytometry offers the possibility of rapid enumeration of parasitemia. It relies on staining the parasite DNA to distinguish between infected and non-infected red blood cell (RBC) populations. Unfortunately, in rodents abundant reticulocyte RNA interferes with the application of the method. This results in time-consuming sample preparation protocols that offer no clear advantage over microscopic counting. We re-evaluated the use of the DNA/RNA discriminating vital fluorochrome acridine orange (AO) for rapid flow cytometric enumeration of parasitemia in rodents. Whole blood from rodents infected with Plasmodium berghei and Plasmodium yoelii was stained with AO and analyzed by flow cytometer. A newly developed two-channel (FL1/FL3) detection method was compared with conventional one-channel (FL1) detection and microscopic counting. The new AO two-channel detection method clearly discriminated between infected and non-infected RBC populations. It showed to be linear above parasitemias of 0.3%. Sample processing time amounted to approximately 5 min. It is shown that AO can be used for rapid, precise, and accurate enumeration of parasitemia in rodents. Due to its ease of handling the method might find widespread application in malaria research.

Separation of live cells in different phases of the cell cycle for gene expression analysis.

Homogeneity of cell populations is a basic requirement for gene expression analyses of the cell cycle, such as those based on microarrays. The most common approach to obtain specific populations is the use of synchronization methods that increase the number of cells representing a certain cell cycle stage. On the one hand, conventional synchronization usually causes undesirable effects. On the other hand, cell separation methods may imply loss of RNA quality, another limiting factor for expression profiling. We describe a new strategy to specifically separate live cells in different phases of the cell cycle (G(1) and G(2)/M) to obtain good quality RNA for gene expression analyses. The experimental design included sorting G(1) and G(2)/M cells with the vital fluorochrome Hoechst 33342, followed by RNA isolation from the sorted cells. Sorted living G(1) and G(2)/M cells, analyzed by immunocytochemistry and laser scanning cytometry, showed strong enrichment. The quality and specificity of the isolated RNA were demonstrated by northern blot. This new approach has many potential applications, such as expression profiling of specific cell populations after eliminating the irrelevant data produced by cells in other stages of the cycle.

Assessment of Escherichia coli B with enhanced permeability to fluorochromes for flow cytometric assays of bacterial cell function.

Flow cytometry has become a choice methodology for microbiological research. However, functional cytometric assays in live bacteria are still limited. This is due, in part, to the cell wall impairing penetration of vital dyes in bacteria, thus imposing permeabilization procedures. These manipulations may affect cell physiology, provoke cell aggregation or lysis, and they are time-consuming. Escherichia coli B strains have been used for mutagenic assays because of an altered lipopolysaccharide that provokes increased membrane permeability. We assessed the use of these strains as possible alternatives for flow cytometric assays to avoid the permeabilization steps. Suspensions of E. coli K-12 (strain AB1157) and E. coli B (strain WP2 uvrA/pKM101, denoted as strain IC188) were stained with several fluorochromes, including fluorescein isothiocyanate, propidium iodide, Nile Red, bis-(1,3-dibutylbarbituric acid) trimethine oxonol, hydroethidine, and dihydro-dichlorofluorescein diacetate, under basal conditions and following permeabilization, impairment of membrane potential, inhibition of dye efflux pump, and oxidative stress. Fluorescent staining of both strains was compared by epifluorescence microscopy and flow cytometry. The E. coli B strain IC188 exhibited more efficient staining with vital fluorochromes than the E. coli K-12 strain AB1157 and maintained a similar membrane potential. In addition, IC188 showed higher sensitivity than AB1157 to reveal oxidative stress when challenged with prooxidants. E. coli B strains may be useful for biochemical and toxicological studies based on flow cytometry and fluorescence microscopy.

Choosing the right sperm for ART: gender and viability selection of spermatozoas by flow cytometry

Objective: Currently the method to choose the spermatozooa to use for ICSI is still a blind manoeuvre, and it can explain failures of fertilization or to allow the transmision of a genetic disease to the offspring due to the selection of a wrong sperm. The aim of this study was to determine the spermatozoa gender and its viability by MicroSort of them with flow cytometry, to enrich the sample by sex and normal spermatozoas, and test the utility of this method to choose the right sperm to use in ART.Design: Prospective and experimental study.Materials/Methods: We analyse by FISH 10 ejaculates samples from sperm donnors. The probes used were: X, Y, 18, and 21, to determine the genetic status of spermatozoas. After that we sorted the remaining sample in a flow cytometry (FACS Vantage, Becton Dickinson, CA, USA). The spermatozoas were stained with the vital fluorochrome bisbenzimide (Hoeschst, Behring, CA, USA), then the sample was passed through the cytometer and sorted for X-bearing spermatozoa and Y-bearing spermatozoa, and chromosomically abnormal, separed and enriched electronically to the diferents fractions. An alliquote of each fraction was again reanalyze by FISH, to check the precision of cytometer procedure. After that the X and Y fractions were stained with fluoresceine diacetate and MicroSorted and enriched by viability.Results: The enrichment by flow cytometry for X, Y and abnormal spermatozoa was almost 90% per group. The viability of spermatozoas in groups X and Y was 95% and 63% in the abnormal group (p <0.05). This sorting and enrichment was confirmed by the second round of FISH.Conclusions: The possibility to diagnose these alterations by FISH of spermatozoa, and further MicroSort by flow cytometry and enrich normal spermatozoas, could avoid the posibility of transmition of some genetic pathology to the offspring and maybe improve the results of some Assisted Reproductive Techniques and sex selection. The flow cytometry is an interesting new technique in human biology of reproduction, that probably will change the current way to prepare the seminal fluid for ART, and probably how to choose the rigth spermatozoa for ICSI procedure. However the safety of these procedures needs to be tested before to use in human clinical practice.Supported by: Reproduccion y Genetica, Hospital Angeles del Pedregal, Universidad La Salle, Biotechnology & Bioengineering Department of Center for Research and Advanced Studies - National Polytechnic Institute (CINVESTAV-IPN), Mexico City. Objective: Currently the method to choose the spermatozooa to use for ICSI is still a blind manoeuvre, and it can explain failures of fertilization or to allow the transmision of a genetic disease to the offspring due to the selection of a wrong sperm. The aim of this study was to determine the spermatozoa gender and its viability by MicroSort of them with flow cytometry, to enrich the sample by sex and normal spermatozoas, and test the utility of this method to choose the right sperm to use in ART. Design: Prospective and experimental study. Materials/Methods: We analyse by FISH 10 ejaculates samples from sperm donnors. The probes used were: X, Y, 18, and 21, to determine the genetic status of spermatozoas. After that we sorted the remaining sample in a flow cytometry (FACS Vantage, Becton Dickinson, CA, USA). The spermatozoas were stained with the vital fluorochrome bisbenzimide (Hoeschst, Behring, CA, USA), then the sample was passed through the cytometer and sorted for X-bearing spermatozoa and Y-bearing spermatozoa, and chromosomically abnormal, separed and enriched electronically to the diferents fractions. An alliquote of each fraction was again reanalyze by FISH, to check the precision of cytometer procedure. After that the X and Y fractions were stained with fluoresceine diacetate and MicroSorted and enriched by viability. Results: The enrichment by flow cytometry for X, Y and abnormal spermatozoa was almost 90% per group. The viability of spermatozoas in groups X and Y was 95% and 63% in the abnormal group (p <0.05). This sorting and enrichment was confirmed by the second round of FISH. Conclusions: The possibility to diagnose these alterations by FISH of spermatozoa, and further MicroSort by flow cytometry and enrich normal spermatozoas, could avoid the posibility of transmition of some genetic pathology to the offspring and maybe improve the results of some Assisted Reproductive Techniques and sex selection. The flow cytometry is an interesting new technique in human biology of reproduction, that probably will change the current way to prepare the seminal fluid for ART, and probably how to choose the rigth spermatozoa for ICSI procedure. However the safety of these procedures needs to be tested before to use in human clinical practice. Supported by: Reproduccion y Genetica, Hospital Angeles del Pedregal, Universidad La Salle, Biotechnology & Bioengineering Department of Center for Research and Advanced Studies - National Polytechnic Institute (CINVESTAV-IPN), Mexico City.

Effect of Relative Humidity on Viability of Primary Conidia ofZoophthora radicans

Four isolates of the entomopathogenic fungusZoophthora radicanswere compared in a laboratory study to evaluate the effect of relative humidity (RH) on duration of primary conidial viability. Primary conidia were showered onto agar-coated glass microscope slides within an enclosed chamber equilibrated to one of five test RH levels (60, 75, 80, 95, or 100%). Target RH levels were achieved by recirculating air through a glycerin/water solution, of controlled specific gravity, contained in a reservoir within the chamber. Conidial samples of each isolate incubated for 5, 10, 30, 60, 120, 180, or 240 min at each RH were removed and inspected using a technique of simultaneous vital fluorochrome staining to determine percentage conidial viability. At 60% RH, isolates did not differ significantly and average viability dropped to less than 10% within the first 60 min. At 75% RH, viability did not change significantly over 4 h. However, average viabilities at 75% RH differed significantly for the four isolates and ranged from 24 to 63%. At 80% RH, viability differed significantly among isolates and declined slowly over time, remaining above 80% for 2 h and above 50% for 4 h. At 95 and 100% RH, average viability was near 95% and did not vary significantly with time or isolate. These data can be used to assist selection of appropriate isolates for biological control.

MACROPHAGE DEPLETION PROMOTES XENOGENEIC HEMATOPOIETIC CHIMERISM

1006 Background: The successful establishment of xenogeneic tolerance through hematopoietic stem cell engraftment is restricted by the rapid disappearance of these cells in the recipient following infusion. Therefore, the goal of these studies was to develop methods to increase survival of pig hematopoietic cell in a xenogeneic recipient. We developed and tested the hypothesis that the mononuclear phagocyte system was responsible for the rapid clearance of infused pig hematopoietic cells. Methods: Balb/c or SCID mice were conditioned with a dose of 3Gy whole body irradiation (WBI), heparin, and cobra venom factor (CVF) on day 0. On various days, mice were injected with medronate-encapsulated liposomes or control blank liposomes, followed by i.v. infusion of miniature swine bone marrow cells. As a means of measuring mononuclear phagocyte function, mice were injected i.v. with FITC-labeled latex beads. The kinetics of bead clearance from the circulation were measured by flow cytometry. To study cell trafficking, swine or autologous bone marrow cells were loaded with a vital fluorochrome. Bone marrow cells (5×107) were then infused i.v. The kinetics of the disappearance of infused cells from the circulation and the trafficking through the spleen and into the bone marrow were determined by flow cytometry. Hematopoietic cells were recovered from the bone marrow at various times and CFU and LTC-IC analyses were performed. Results: Medronate-encapsulated liposomes are non-toxic in mice at levels that deplete mononuclear phagocytes. Depletion of mononuclear phagocytes in Balb/c mice as well as in SCID mice increases the accumulation pig hematopoietic cells in the bone marrow by 10-fold when measured 24hr post bone marrow cell infusion. Following phagocyte depletion, the accumulation of pig cells in the bone marrow of Balb/c mice is approximately two-fold less than that observed following autologous bone marrow cell infusion. LTC-IC assays indicate an increased presence of pig progenitors in the bone marrow of mice which were treated pre- and post- bone marrow infusion with medronate-liposomes. Experiments to investigate the effect of phagocyte depletion on pig cell chimerism in primates are in progress. Conclusion: We have found that mononuclear phagocytes play a major role in the elimination of pig hematopoietic cells in mice. Depletion of mononuclear phagocytes in mice leads to a significant increase in pig hematopoietic chimerism and this liposome treatment is not toxic to pig hematopoietic progenitors. This work was supported by BioTransplant, Inc.

Controlled cultivation of Alexandrium minutum and [ 33P] orthophosphate cell labeling towards surface adhesion tests

We describe a protocol for preparing cultures of the PSP (paralytic shellfish poisoning) dinoflagellate Alexandrium minutum, towards subsequent studies of cell adhesion onto artificial substrates. First, phenotypic uniformity of the strain used and reproducibility of the standard growth profile are obtained by optimising key parameters. Batches of A. minutum at mid-exponential proliferation phase are radiolabeled with 33P-containing medium in order to later quantify cell adhesion. A mortality corrective index is applied to the latter, using the vital fluorochrome acridine orange, i.e. dead cells show no nuclear incorporation under epifluorescence microscopy.

Characterization of cell death induced by 2-methoxyethanol in CD-1 mouse embryos on gestation day 8.

Cell death was analyzed in neurulating mouse embryos after in vivo doses of 2-methoxyethanol (2-ME) that produce anterior neural tube defects. Characterization of 2-ME-induced cell death was performed by evaluating: (1) vital fluorochrome staining in whole embryos applying confocal laser scanning microscopy; (2) characteristics of cell debris in conventional histological sections revealed by light microscopy; and (3) Apoptag in situ immunohistochemical staining for apoptosis using light microscopy. Methods for quantification of cell death identified by these three techniques were explored using computerized image analysis. Physiological cell death in control embryos primarily occurred in the neural crest region during neural fold elevation. Embryos exposed to 2-ME had expanded areas of cell death in the neural crest and also new areas of cell death in medial regions of the anterior neural tube. Both physiological and 2-ME-induced embryonic cell death had morphological, immunohistochemical, and fluorochrome staining characteristics of apoptosis. When fluorescence data from confocal microscopic analysis of vital fluorochrome-stained embryos were analyzed, a dose-dependent increase was found in embryos exposed to 2-ME. Similar results were obtained when cell death was analyzed in either conventional histological sections or sections prepared for immunohistochemical detection of apoptosis. The cell death data obtained in this study correlate with previously observed near-term malformation rates, suggesting that a quantitative relationship exists between 2-ME-induced embryonic cell death and neural tube defects.

Characterization of cell death induced by 2‐methoxyethanol in CD‐1 mouse embryos on gestation day 8

Cell death was analyzed in neurulating mouse embryos after in vivo doses of 2-methoxyethanol (2-ME) that produce anterior neural tube defects. Characterization of 2-ME-induced cell death was performed by evaluating: (1) vital fluorochrome staining in whole embryos applying confocal laser scanning microscopy; (2) characteristics of cell debris in conventional histological sections revealed by light microscopy; and (3) Apoptag™ in situ immunohistochemical staining for apoptosis using light microscopy. Methods for quantification of cell death identified by these three techniques were explored using computerized image analysis. Physiological cell death in control embryos primarily occurred in the neural crest region during neural fold elevation. Embryos exposed to 2-ME had expanded areas of cell death in the neural crest and also new areas of cell death in medial regions of the anterior neural tube. Both physiological and 2-ME-induced embryonic cell death had morphological, immunohistochemical, and fluorochrome staining characteristics of apoptosis. When fluorescence data from confocal microscopic analysis of vital fluorochrome-stained embryos were analyzed, a dose-dependent increase was found in embryos exposed to 2-ME. Similar results were obtained when cell death was analyzed in either conventional histological sections or sections prepared for immunohistochemical detection of apoptosis. The cell death data obtained in this study correlate with previously observed near-term malformation rates, suggesting that a quantitative relationship exists between 2-ME-induced embryonic cell death and neural tube defects. Teratology 58:231–240, 1999. © 1998 Wiley-Liss, Inc.

Actomyosin-based motility of endoplasmic reticulum and chloroplasts in Vallisneria mesophyll cells

Intracellular localization and motile behaviour of the endoplasmic reticulum (ER), plastids and mitochondria were studied in living mesophyll cells of Vallisneria using the vital fluorochrome 3,3'-dihexyloxacarbocyanine iodide (DIOC6(3)). In quiescent cells, the ER was composed of a three-dimensional network of tubular and lamellar elements. Chloroplasts were distributed evenly throughout the cell periphery and appeared embedded within the ER network. The ER network was relatively stationary, with the exception of rare motile episodes occurring as movement of tubular ER strands and adjacent areas of the polygonal network in localized areas of the cell. During experimental induction of streaming, most of the lamellar ER elements transformed into tubules and together with the chloroplasts they began to translocate to the anticlinal walls to establish the circular streaming around the circumference of the cell. Microwave-accelerated fixation followed by immunofluorescence revealed an hitherto unknown phase of actin reorganization occurring within the cells and most interestingly at the surface of the chloroplasts during streaming induction. Myosin was localized in an ER-like pattern in quiescent as well as in streaming cells, with bright fluorescent label localized on mitochondria and proplastids. In addition, myosin label appeared on the surface of the chloroplasts, preferentially in streaming mesophyll cells. Motile activities were impeded by the actin-depolymerizing drug cytochalasin D (CD), the thioreagent N-ethylmaleimide (NEM), and thapsigargin, an inhibitor of the ER-Ca(2+)-ATPase. These inhibitors also interfered with the integrity of actin filaments, the intracellular distribution of myosin and calcium-homeostasis, respectively. These effects suggested an obligate association of at least one type of myosin with the membranes of ER and smaller organelles and are consistent with the appearance of another type of myosin on the chloroplast surface upon streaming induction.

Vital fluorochromes as tracers for fungal growth studies.

Eight fluorescent dyes were tested for staining the spores or mycelia of six fungi and for their translocation into new growth when the preloaded spores or mycelia were incubated on agar coated coverslips. The dyes studied were Cellufluor, Nile red, fluorescein diacetate (FDA), carboxyfluorescein diacetate (CFDA), chloromethylfluorescein diacetate (CMFDA), aminochloromethyl coumarin (CMAC), and the carbocyamines DiIC18(3) and DiOC18(3). The fungi on which the dyes were tested included Botrytis cinerea, Fusarium oxysporum f.sp. lycopersici, Idriella bolleyi, Pythium oligandrum, Sclerotium cepivorum and Trichoderma harzianum. Most of the fluorochromes gave good initial staining of mycelia or spores; however, FDA fluorescence faded rapidly during excitation, making it impractical for use. Also, the spores and mycelia of B. cinerea and T. harzianum sometimes gave weak fluorescence with Nile red, and the spores and mycelia of I. bolleyi gave unusually weak fluorescence with Cellufluor. There were other variations of staining among the different dye/fungus combinations, but each fungus showed strong fluorescence at least one dye. Cellufluor, CMFDA, CMAC and, to a lesser extent, CFDA and Nile red, were efficiently translocated into new growth from preloaded spores or mycelia, whereas FDA, DiIC18(3) and DiOC18(3) were not. The extent of translocation ranged from 0.1 to 1.2 mm in germ tubes arising from spores, and from 0.9 to 9.2 mm in mycelia extending from dye-loaded agar blocks. The findings suggest that fluorescent dyes could be used as markers or tracers in studies of fungal growth and differentiation.

An interconnected plastidom inAcetabularia: Implications for the mechanism of chloroplast motility

In the unicellular green algaAcetabularia, the vital fluorochrome 3,3′-dihexyloxacarbocyanine (DiOC6) readily accumulates in chloroplasts and mitochondria at low concentrations, suboptimal for the visualization of the endoplasmic reticulum (ER). These organelles align along motility tracks and partially obscure each other, resulting in the loss of image information in conventional fluorescence microscopy. However, superior imaging of organelles was achieved by confocal laser scanning microscopy, which was particularly evident in areas where mitochondrial profiles overlap with chloroplasts. In addition to the tubular mitochondria, a new type of tubular membrane profiles was discovered inAcetabularia which connects the chloroplasts with each other. These tubules may either form short bridges or may stretch over hundreds of micrometers before connecting to the next chloroplast. Because staining intensity, size and overall shape of mitochondria and the connecting membrane tubules were very similar, pharmacological treatments have been applied to differentiate more clearly between the two compartments. Inhibitors of mitochondrial function are shown here to affect mitochondrial shape but not that of the chloroplast tubules. Finally, electron microscopic analysis of thin sectioned materials revealed long tubular emanations from the chloroplasts proving their plastidal origin. The function of these hitherto unknown plastidal membrane tubules is not known, but their behaviour suggests that they interact with the cytoskeleton and effectively modify chloroplast behaviour.

Effect of extracorporeal membrane oxygenation on neutrophil function in neonates.

To study the effect of long-term extracorporeal membrane oxygenation (ECMO) support on neutrophil function. A prospective, clinical investigation. A pediatric intensive care unit. Four groups of patients: ECMO group 1, newborns after 1 day of ECMO support (n = 10); ECMO group 2, newborns after 5 days of ECMO support (n = 6); group 3, normal newborns (n = 20); group 4, normal adults (n = 30). Two mL of heparinized blood was obtained from patients in each group. A modification of the Smith and Rommel technique was used to measure neutrophil phagocytosis and killing utilizing live Candida tropicalis as the test organism. Neutrophils were incubated for 90 mins in normal adult serum with live Candida. Viability of Candida after phagocytosis was tested by vital fluorochrome staining. Phagocytic index (the number of neutrophils with intracytoplasmic Candida divided by the total neutrophils) and candidicidal ratio (neutrophils with dead Candida divided by total neutrophils with Candida) were determined daily. Neutrophils from ECMO group 1 (day 1) and ECMO group 2 (day 5) patients had significantly higher phagocytosis indices (72.8 +/- 20 and 76 +/- 18) and candidicidal ratios (0.15 +/- 0.1 and 0.16 +/- 0.09) compared with neutrophils from group 3 patients (normal neonates) (64 +/- 7 and 0.06 +/- 0.04). The phagocytosis indices were significantly lower in neutrophils from ECMO group 1 (day 1) and ECMO group 2 (day 5) patients compared with group 4 (adults) patients (86 +/- 9). However, the candidicidal ratios in neutrophils from ECMO groups 1 and 2 (ECMO day 1 and day 5) patients were equal to that value in group 4 (adults) (0.10 +/- 0.04). ECMO support for 5 days (ECMO group 2 vs. group 1) did not significantly change either the phagocytosis index or candidicidal ratio. Phagocytosis and intracellular killing by neutrophils of ECMO-supported neonates were significantly greater than those values found in normal newborns. ECMO support for 5 days produced no significant changes in neutrophil phagocytosis or killing.

Early steps of the P-glycoprotein expression in cell cultures studied with vital fluorochrome

This study shows that flow cytometry analysis of the rate of fluorochrome Rh123 efflux may be used for detection of cells at initial steps of P-glycoprotein expression and of minor subpopulations of multidrug-resistant (MDR) variants in human cell lines. This method also evaluates the fraction of low-level MDR cells among peripheral blood leukocytes of patients with chronic myeloid leukemia. The alterations in Pgp function were revealed in rat hepatoma cells after short treatment with colchicine.