Use Of Calcein Research Articles

Efficacy of fluorescent calcein mark retention and identification in endangered Colorado pikeminnow Ptychocheilus lucius

AbstractDistinguishing hatchery‐reared fishes from wild conspecifics can be required to quantify the success of augmentation programmes. This study estimated the probability of identifying calcein‐marked, hatchery‐reared Colorado pikeminnow Ptychocheilus lucius Girard from external and internal structures. Both control and marked fish held in the laboratory were correctly identified 80% of the time after 300 days. A fluorometer was more accurate 180 days post‐marking, whereas visual observation was more discriminating at 300 days. There were no differences in detection rates among structures in laboratory fish, and for most structures of fish held outdoors, detection rates were <20%. Overall, a strong negative effect of light on mark persistence was observed. Also, an initial positive effect of fish size on mark fluorescence and a negative effect of fish growth were detected. These results suggest the potential use of calcein as a batch‐marking tool would be aided by future studies that better quantify light exposure and calcein mark persistence in both field and experimental settings.

Calcein Staining as a Tool to Investigate Coccolithophore Calcification

Despite the oceanographic and geological significance of coccolithophores, the cellular mechanisms that underlie the intracellular production and subsequent secretion of their CaCO3 coccoliths remain poorly understood. Tools for labelling coccoliths and coccospheres in order to track their production would be of great value. We therefore evaluated the use of calcein, a derivative of fluorescein, as a method to fluorescently label coccoliths. The calcein method readily labelled pre-existing coccospheres in a range of coccolithophore species, including diploid and haploid life history phases, without compromising the coccolith structure. Calcite staining was verified though epifluorescence and confocal microscopy, and both stained and unstained cells and coccoliths were readily distinguished using flow cytometry. The fluorescence of stained coccoliths was retained for > 3 d allowing us to confirm the polar secretion of coccoliths by distinguishing pre-existing coccoliths from the accumulation and distribution of non-fluorescent coccoliths produced after calcein exposure. The calcein treatment had no significant effect on photosynthetic physiology, external calcite morphology, or growth rates of the cells over an 8 d period. The calcein staining method therefore represents a simple non-invasive, non-toxic optical technique to ‘tag’ calcium carbonate coccoliths and track their production in response to environmental manipulations or pharmacological treatments. Moreover, calcein staining of the coccosphere allowed for heterogenous patterns of calcification, growth, and cell division to be detected in a population of cells. This is the first description of the use of calcein to stain the biomineral structures of calcifying phytoplankton and this approach has the potential to be applied to detailed cytological investigations as well as high-throughput analysis of cultured cells or field populations.

Potential impacts of blooms of the toxic dinoflagellate Karenia brevis on the growth, survival and juvenile recruitment of the non-native green mussel Perna viridis in southeastern United States

Red tide blooms formed by Karenia brevis are frequent along the Gulf coast of Florida and it is unclear what tolerance the green mussel Perna viridis, a recently introduced species to coastal waters, has toward these events. Established populations of P. viridis were monitored along the coastal waters of Estero Bay, Florida before, during and following two consecutive red tide blooms to assess the potential effects on growth, survival and juvenile recruitment. Upon onset of the bloom, growth rates fell from 6 to 10 mm month−1 (March 2011–November 2011) to less than 3 mm month−1. In the succeeding years, K. brevis blooms were present, and average growth of individually tagged mussels remained below 3 mm month−1. During growth monitoring the use of calcein as an internal marker was tested with positive staining results and no observed effect on growth or survival. In March 2012, following the first red tide bloom, a population-wide mortality event was observed. Following this event, increased mortality rates were observed with peaks during onset of the bloom in the fall of 2012 and 2013. Juvenile recruitment was also limited during years in which blooms persisted into the spring spawning period suggesting gamete and/or larval sensitivity to K. brevis. Although it cannot be conclusively determined that the cause of reduced growth and survival is due to red tide events, the parallels observed suggest that K. brevis is a factor in the observed changes in population structure.

A novel antimicrobial peptide, scolopendin, from Scolopendra subspinipes mutilans and its microbicidal mechanism.

A novel antimicrobial peptide (AMP) was identified from the centipede Scolopendra subspinipes mutilans by RNA sequencing, and the amino acid sequences predicted from the sequenced mRNAs were compared with those of known AMPs. We named this peptide scolopendin, according to its origin, and investigated the molecular mechanisms underlying its antimicrobial activity. Our findings showed that scolopendin had antimicrobial activity against several pathogenic microorganisms, but did not produce hemolysis of human erythrocytes. In addition, disturbances in the cell membrane potential, induction of potassium release from the cytosol, and increased membrane permeability of the microbes Candida albicans and Escherichia coli O157 were detected by the use of 3,3'-dipropylthiacarbocyanine iodide [DiSC3(5)] dye, potassium leakage assay, and propidium iodide influx assay, respectively, following scolopendin treatment. Further evidence to support the membrane-targeted action of scolopendin was obtained using artificial liposomes as models of the cell membrane. Use of calcein and FITC-labeled dextran leakage assays from scolopendin-treated giant unilamellar vesicles and large unilamellar vesicles showed that scolopendin has a pore-forming action on microbial membrane, with an estimated pore radius of 2.3-3.3nm. In conclusion, scolopendin is a novel and potent AMP with a membrane-targeted mechanism of action.

Fracture characterization in cancellous bone specimens via surface difference evaluation of 3D registered pre- and post-compression micro-CT scans

In recent years, increasingly stringent passenger vehicle safety requirements have led to a renewed interest in the fracture properties of bone. It has been shown that cancellous bone architecture is strongly linked to its overall behavior (Follet et al. 2011; Prot et al. 2015). Micro-fracture mechanisms have been resolved by time-consuming direct microscopy (Prot et al. 2012) or by the use of calcein (Lambers et al. 2014; Hernandez et al. 2014). Furthermore, the application of CT scanners, along with the development of registration algorithms, has allowed separated portions of fractured specimen to be registered to the pre-compression scan so as to quantify the difference between 3D shapes as a mean to characterize the fracture behavior (Tassani & Matsopoulos 2014). However, this method is operator-dependent in the case of multiple fracture zone identification and requires sufficient deformation of the specimen to obtain distinct registration sets. In addition, built-in micro-compression testers, developed by CT scanner manufacturers, are limited to loading at low levels of strain rate, which does not allow measurement over a range that is representative of daily life. The goal of this study was to identify distinct fracture patterns based on micro-CT scans of cancellous bone specimens, loaded over a large range of strain rates, without the need for specimens that have broken into separate pieces.

Growth rate variation of the stalked barnacle Pollicipes pollicipes (Crustacea: Cirripedia) using calcein as a chemical marker

This study describes the use of calcein as a chemical tagging methodology to estimate growth rate variation of the stalked barnacle Pollicipes pollicipes, an ecologically important intertidal species and economic resource, in SW Portugal. Calcein tagging had a high success rate (94%) in marking both juvenile and adult barnacles for a period of 2.5 months, providing a valuable method for obtaining reliable data in growth studies of P. pollicipes. Growth rate decreased with barnacle size and was highly variable amongst individuals, particularly in smaller barnacles. No effect of shore level on barnacle growth was detected. Growth rates were higher in smaller juvenile barnacles, peaking at a 1.1-mm monthly increment in rostro-carinal length (RC) for individuals with RC=5 mm, and decreased with barnacle size (monthly growth rates of 0.5 mm for adult barnacles with RC~12.5 mm). Growth rates observed in adults with commercial interest (RC ≥ 18 mm) was < 0.25 mm per month. The advantages of tagging P. pollicipes with calcein were the possibility of mass marking individual barnacles of different size cohorts within a short period (less than 1 day of manipulation); and reduced time of fieldwork, which is very important because this species inhabits very exposed rocky shores.

Use of calcein and alizarin red S for immersion marking of black rockfish Sebastes schlegelii juveniles

Black rockfish Sebastes schlegelii juveniles (30–40 mm total length) were immersed in a range of calcein (CAL) solutions at concentrations ranging from 50 to 250 mg/L and alizarin red S (ARS) solutions at concentrations ranging from 100 to 500 mg/L in filtered seawater (salinity 30) for 24 h. Fluorescent marks were detected in otoliths (sagittae, asteriscus), scales, fin rays (dorsal, pectoral, ventral, anal, and caudal fin rays), and fin spines (dorsal, ventral, and anal fin spines) after a 60-d growth experiment. With the exception of 50–100 mg/L CAL, acceptable marks were produced in the otoliths and fin spines by all concentrations of CAL and ARS. In particular, marks were clearly visible under normal light in the sagittae, asteriscus, and fin spines of fish immersed in 200–500 mg/L, 300–500 mg/L, and 200–500 mg/L ARS, respectively. Scales and fin rays had acceptable marks at much higher concentrations (≥50 mg/L CAL, ≥300 mg/L ARS for scales and ≥50 mg/L CAL, ≥200 mg/L ARS for fin rays). The mark quality was highest (i.e., acceptable marks were observed in all sampled structures after immersion marking) in fish immersed in 150–250 mg/L CAL or 300–500 mg/L ARS. In addition, there was no significant difference in survival and growth of marked fish compared with controls 60 d post-marking ( P >0.05).

External fluorescence retention of calcein‐marked juvenile brown trout Salmo trutta raised in natural and artificial environments

The fluorescence retention and intensity of juvenile brown trout Salmo trutta marked during their first summer were monitored in a hatchery and in four natural streams. A handheld detector was used for direct examination. In the hatchery, three marking treatments (T) were compared: 3·5 min in a 0·5% calcein solution (T0·5-3·5), 7 min in a 0·5% calcein solution (T0·5-7) and 3·5 min in a 1% calcein solution (T1-3·5). The fish were raised indoors for 11 months and then outdoors until 18 months. The fluorescence retention rate was 100% in all treatments at 11 months, although T1-3·5 showed the highest mean fluorescence intensity, followed by T0·5-7 and T0·5-3·5. The fluorescence intensity was not correlated with the final total length (L(T)) of the fish in two treatments, although it significantly decreased with increasing L(T) in T1-3·5. At 18 months, <30% of the fish were still slightly fluorescent, suggesting a negative effect of sunlight exposure. In stream studies, the fluorescence intensity did not significantly differ according to final L(T); an overall mean ± s.d. retention rate of 70·7 ± 26·6% was measured at 12 months with a decrease to 48·6 ± 24·6% at 24 months. Significant differences amongst streams and within reaches of the same stream were observed. Because of a significant positive effect of the shading index on the fluorescence intensity, the use of calcein should be restricted to streams unexposed to direct sunlight. Consequently, the marking method would be reliable for 1 year monitoring studies in shaded streams.

Detection of Volume Changes in Calcein-Stained Cells Using Confocal Microscopy

Calcein is an intracellular fluorescent probe that has been used as an indicator of cell volume in several previous studies. These studies have reported two different fluorescence responses depending on the optical setup used to collect the data: wide-field microscopy has resulted in a decrease in fluorescence upon cell shrinkage, whereas confocal microscopy has been shown to yield the opposite result. In this short communication, we have investigated the effect of optical setup on detection of cell volume changes in calcein-stained endothelial cells. A confocal microscope was used to collect the fluorescence data, and the pinhole diameter was varied in order to examine the effects of optical section thickness on fluorescence response. For large pinhole diameters - which correspond to relatively thick optical sections - fluorescence intensity decreased when cells were induced to shrink. In contrast, for small pinhole diameters the fluorescence intensity increased with cell shrinkage. The transition between these two types of fluorescence responses occurred when using a pinhole diameter of 285μm, which corresponds with an optical section thickness slightly less than the height of the cells. Our results have implications for the design and interpretation of experiments involving the use of calcein as a cell volume indicator.

Use of calcein to estimate and validate age in juveniles of the winged pearl oysterPteria sterna

Determining age is an important step when assessing growth, mortality, and yield of cultivated and wild populations, but studies linking shell growth marks in the pearl oyster Pteria sterna with the age of individuals are lack- ing. Thirty juveniles (20.0 ± 1.2 mm shell height), collected from a winter spatfall, were marked with the fluorochrome calcein and kept in the field in culture containers. After day 16, the juveniles were cleaned and their shells cut along the sagittal axis to determine periodicity of micro growth bands formed in the inner shell layers and to estimate age. During this trial, fluorescent calcein marking succeeded in individuals larger than 20 mm shell height; these formed an average of 15 micro growth bands over the 16 days, representing 1 band per day. The marker created a wide fluorescent band containing three micro growth marks, suggesting that calcein was incorporated into the shell over the first three days. The use of calcein was found to be an accurate method for validating the micro growth band frequency of formation in P. sterna juveniles, which in turn can help to estimate age.

Optimising chemical marking techniques for Australian bass, Macquaria novemaculeata, fry and fingerlings prior to restocking

Many stocking programs are performed without any subsequent assessments to determine the survival of the stocked fish, often due to the difficulties in distinguishing stocked from wild fish. A series of optimisation trials were undertaken to determine the suitability of calcein (2,4-bis-[N,N′–di(carbomethyl)-aminomethyl]fluorescein) for chemically marking hatchery-reared Australian bass, Macquaria novemaculeata (Steindachner), fry and fingerlings. The technique was optimised by trialling immersion times for salt (2.5 or 5 min), calcein concentrations (0.5 or 1%) and calcein immersion times (5 or 10 min). A general-purpose modulated probe fluorometer was used as a non-lethal detection method to establish mark retention and distinguish between calcein-marked and unmarked fish. Five minutes of saline immersion, followed by 10 min of calcein immersion at a calcein concentration of 1% was the optimal marking method. Growth and survival of fish was not affected as a result of any of the calcein-marking methods. It is therefore effective to calcein-mark Australian bass fry and fingerlings before stocking and to non-lethally detect marked individuals using a field-portable meter to assist in the assessment of stocking programs. Findings from this study may also reflect the potential use of calcein to chemically mark fry and fingerlings of other fish species.

New insights into the mode of action of ultradeformable vesicles using calcein as hydrophilic fluorescent marker

Whether ultradeformable vesicles pass intact through the stratum corneum and can promote the transdermal absorption of any substance remain open questions. This paper presents different experimental approaches, based on the use of calcein as hydrophilic fluorescent marker, to probe the physicochemical and pharmacokinetic characteristics of these vesicles. Ultradeformable membranes made from natural phosphatidylcholine and sodium cholate were found to be highly permeable to calcein, as a result of the permeabilizing effects of sodium cholate and ethanol. In vitro skin permeation and in vivo transdermal (percutaneous) absorption studies were performed using hairless mice. Both studies indicated that deformable vesicles reduce the transdermal flux of calcein, when compared to a solution containing or not sodium cholate and ethanol. The data support the model that the transdermal absorption of calcein from deformable vesicles is controlled by the release of the drug from the formulation deposited onto the skin surface. Importantly, fluorescence measurements of the receptor fluid of the Franz diffusion cell after addition of Co 2+ quencher revealed that permeated calcein exists essentially under the non-encapsulated form. In conclusion, our results argue against the model that deformable vesicles would carry hydrophilic drugs across the skin and act as a sustained release system in deep tissues.

The intracellular iron sensor calcein is catalytically oxidatively degraded by iron(II) in a hydrogen peroxide-dependent reaction

The fluorescent metal chelating dye calcein is used to obtain an estimate of cellular iron levels and to measure the kinetics of the entry of chelators and chelating drugs into cells. Under reducing conditions in the presence of ascorbic acid, such as that would be present in the cell, the Fe(II)–calcein complex was rapidly formed with a rate constant of 3×10 5 M −1 s −1. A slower iron-dependent catalytic degradation of calcein also occurred that resulted in the formation of a non-fluorescent calcein product. The Fe(II)-catalyzed degradation of calcein was largely, but not completely, prevented by catalase. Electron paramagnetic resonance spin trapping experiments showed that the Fe(II)–calcein complex promoted formation of hydroxyl or a hydroxyl radical-like species. Together these results indicated that Fe(II) catalyzed the degradation of calcein through both hydrogen peroxide, and to a lesser extent, non-hydrogen peroxide-dependent pathways. The iron–calcein complexes that were responsible for the degradation of calcein were likely high valence oxidizing iron-oxo species such as perferryl or ferryl complexes that were redox cycled by ascorbic acid. Thus, the use of calcein as an intracellular iron-sensing indicator may yield misleading results due to its degradation under certain conditions.

Fluorescence Correlation Spectroscopy: A New Tool for Probing the Microenvironment of the Internal Space of Organelles

A possible use of fluorescence correlation spectroscopy (FCS) was demonstrated for the investigation of the microenvironment of the internal space of organelles in living cells. As its first step, we attempted to measure the viscosity of the aqueous phase in endosomes of cultured bovine aortic cells by evaluating fluorescence autocorrelation functions (FAFs) of ingested calcein molecules in endosomes. Multi-component analysis of measured FAFs yielded the average diffusion coefficient of 1.5 × 10-6 cm2s-1, about 45% smaller than that in water. We concluded that the viscosity of endosomal aqueous phase is approximately 2.2-fold of that of water. In addition, we discussed the possible use of calcein as a pH indicator based on the measurement of its pH-dependent fluorescent properties using FCS.

Calcein as a Fluorescent Probe for Ferric Iron: APPLICATION TO IRON NUTRITION IN PLANT CELLS

The recent use of calcein (CA) as a fluorescent probe for cellular iron has been shown to reflect the nutritional status of iron in mammalian cells (Breuer, W., Epsztejn, S., and Cabantchik, Z. I. (1995) J. Biol. Chem. 270, 24209-24215). CA was claimed to be a chemosensor for iron(II), to measure the labile iron pool and the concentration of cellular free iron(II). We first study here the thermodynamic and kinetic properties of iron binding by CA. Chelation of a first iron(III) involves one aminodiacetic arm and a phenol. The overall stability constant log beta111 of FeIIICAH is 33. 9. The free metal ion concentration is pFeIII = 20.3. A (FeIII)2 CA complex can be formed. A reversible iron(III) exchange from FeIIICAH to citrate and nitrilotriacetic acid is evidenced when these ligands are present in large excess. The kinetics of iron(III) exchange by CA is compatible with metabolic studies. The low reduction potential of FeIIICAH shows that the ferric form is highly stabilized. CA fluorescence is quenched by 85% after FeIII chelation but by only 20% using FeII. Real time iron nutrition by Arabidopsis thaliana cells has been measured by fluorimetry, and the iron buffer FeIIICAH + CA was used as source of iron. As a siderophore, FeIIICAH promotes cell growth and regreening of iron-deficient cells more rapidly than FeIIIEDTA. We conclude that CA is a good chemosensor for iron(III) in cells and biological fluids, but not for Fe(II). We discuss the interest of quantifying iron buffers in biochemical studies of iron, in vitro as well as in cells.

Use of calcein and SYTO-13 to assess cell cycle phases and osmotic shock effects on E. coli and Staphylococcus aureus by flow cytometry

The fluorochromes SYTO-13 and calcein were tested by flow cytometry for their ability to assess the cell phases and the effects of osmotic shock in E. coli and S. aureus after exposure to NaCl and sucrose. Results were validated by viable plate count. Flow cytometry gave quicker results and more accurate information about intermediate states and population heterogeneity of S. aureus and E. coli than viable plate counts. SYTO-13 was an excellent marker for total counts and calcein can be used to assess esterase activity in S. aureus.

Use of Calcein as a Fluorescent Marker for Elasmobranch Vertebral Cartilage

Abstract The effectiveness of calcein as a fluorescent marker of elasmobranch vertebrae was examined in the nurse shark Ginglvmostotna cirratum. Intramuscular injection of calcein at a standard dosage of 25 mg/kg body weight (BW) produced distinct fluorescent marks in nurse shark vertebrae; however, it also induced physiological stress and mortality. Lower dosages of 10 and 5 mg/kg BW also produced chemical marks suitable for age validation but were not associated with animal death. These data suggest that calcein dosage levels recommended for teleost age validation may be toxic to elasmobranchs. Future studies should focus on the toxicity of lower-dosage calcein injections.

Spectrophotometric micro method for measurement of dialyzable calcium by use of cresolphthalein complexone and continuous-flow analysis.

We adapted a method for dialyzable calcium from fluorometric detection by use of calcein to a more specific spectrophotometric determination with cresolphthalein complexone. The reagents are available commercially and perform satisfactorily with respect to noise level, drift, stability, and sensitivity. Construction of the continuous-flow manifold with commercially available components (injection blocks, coils, and dialyzer) and the use of a 9-mm pathlength flow cell in an AutoAnalyzer I colorimeter have permitted a sampling rate of 70/h, and decreased the volume of serum required to 130 microL. A comparison of 71 sera analyzed by the present and the calcein method gave means of 1.39 (SD 0.14) and 1.39 (SD 0.13) mmol/L, respectively. The regression equation was: present method = 1.016 calcein--0.022 mmol/L (r = 0.97). The CV for the new method, as determined from 46 randomized duplicate analyses, was < 1%. The reference interval, as evaluated from results for 93 different individuals, was 1.26-1.43 mmol/L. We conclude that our methods is an improvement with respect to noise level, drift, specificity, detectability, and more general availability of instrumentation. Moreover, the smaller sample volume required makes possible the routine measurement of dialyzable calcium in pediatric samples.

Investigation of the Use of Calcein in the Ultramicro Fluorometric Determination of Calcium in Serum

Abstract Calcium in serum is commonly determined fluorometrically with calcium-calcein as the fluorescent complex. A method is presented for analyzing calcein to determine the optimal concentration for both greatest linearity and greatest reproducibility. Serum calcium as determined with the "UNOPETTE" system is compared with results obtained by use of a Technicon SMA 12/60.

Determination of calcium in biological material: The use of calcein as an indicator in the edta titration

The EDTA titration of calcium using the change in fluorescence of Calcein indicator under ultraviolet illumination at the end-point was very satisfactory for essentially pure calcium chloride solutions. In order to use this method for biological samples, it was found best to separate the calcium by an oxalate precipitation at a pH of 4.7, convert the calcium oxalate to calcium carbonate, and dissolve the calcium carbonate in hydrochloric acid.