A Medium-Throughput System for In Vitro Oxidative Stress Assessment in IPEC-J2 Cells.

The feed industry continuously seeks new molecules with antioxidant capacity since oxidative stress plays a key role in intestinal health. To improve screening of new antioxidants, this study aims to set up an assay to assess oxidative stress in the porcine small intestinal epithelial cell line IPEC-J2 using plate-reader-based analysis of fluorescence. Two oxidants, H2O2 and menadione, were tested at 1, 2 and 3 mM and 100, 200 and 300 µM, respectively. Trolox (2 mM) was used as the reference antioxidant and the probe CM-H2DCFDA was used to indicate intracellular oxidative stress. Cell culture, reactive oxygen species (ROS) production and assessment conditions were optimized to detect a significant ROS accumulation that could be counteracted by pre-incubation with trolox. Menadione (200 µM) reproducibly increased ROS levels, H2O2 failed to do so. Trolox significantly decreased intracellular ROS levels in menadione (200 µM)-exposed cells in a consistent way. The system was further used to screen different concentrations of the commercially available antioxidant ELIFE®. Concentrations between 100 and 200 ppm protected best against intracellular ROS accumulation. In conclusion, the combination of CM-H2DCFDA fluorescence analysis by a plate-reader, trolox as a reference antioxidant and 200 µM of menadione as a stressor agent, provides a replicable and reliable medium-throughput setup for the evaluation of intracellular oxidative stress in IPEC-J2 cells.

Hoechst-tagged Fluorescein Diacetate for the Fluorescence Imaging-based Assessment of Stomatal Dynamics in Arabidopsis thaliana.

In plants, stomata regulate water loss through transpiration for plant growth and survival in response to various environmental stressors; and simple methods to assess stomatal dynamics are needed for physiological studies. Herein, we report a fluorescence-imaging-based method using fluorescein diacetate tagged with Hoechst 33342, a nuclear staining chemical probe (HoeAc2Fl) for the qualitative assessment of stomatal dynamics. In our method, the stomatal movement is inferred by simple monitoring of the fluorescence intensity in the nucleus of the stomata.

Liposomal membrane permeability assessment by fluorescence techniques: Main permeabilizing agents, applications and challenges.

Liposomes are lipid vesicles made of one or multiple lipid bilayers surrounding an internal aqueous core. They are broadly employed as models to study membrane structure and properties. Among these properties, liposome membrane permeability is crucial and widely assessed by fluorescence techniques. The first part of this review is devoted to describe the various techniques used for membrane permeability assessment. Attention is paid to fluorescence techniques based on vesicle leakage of self-quenching probes, dye/quencher pair or cation/ligand pair. Secondly, the membrane-active agents inducing membrane permeabilization is presented and details on their mechanisms of action are given. Emphasis is also laid on the intrinsic and extrinsic factors that can modulate the membrane permeability. Hence, a suitable liposomal membrane should be formulated according to the aim of the study and its application.

Assessment of the degradation rates and effectiveness of different coated Mg-Zn-Ca alloy scaffolds for in vivo repair of critical-size bone defects.

Surgical repair of bone defects remains challenging, and the search for alternative procedures is ongoing. Devices made of Mg for bone repair have received much attention owing to their good biocompatibility and mechanical properties. We developed a new type of scaffold made of a Mg-Zn-Ca alloy with a shape that mimics cortical bone and can be filled with morselized bone. We evaluated its durability and efficacy in a rabbit ulna-defect model. Three types of scaffold-surface coating were evaluated: group A, no coating; group B, a 10-µm microarc oxidation coating; group C, a hydrothermal duplex composite coating; and group D, an empty-defect control. X-ray and micro-computed tomography(micro-CT) images were acquired over 12 weeks to assess ulnar repair. A mechanical stress test indicated that bone repair within each group improved significantly over time (P < 0.01). The degradation behavior of the different scaffolds was assessed by micro-CT and quantified according to the amount of hydrogen gas generated; these measurements indicated that the group C scaffold better resisted corrosion than did the other scaffold types (P < 0.05). Calcein fluorescence and histology revealed that greater mineral densities and better bone responses were achieved for groups B and C than for group A, with group C providing the best response. In conclusion, our Mg-Zn-Ca-alloy scaffold effectively aided bone repair. The group C scaffold exhibited the best corrosion resistance and osteogenesis properties, making it a candidate scaffold for repair of bone defects.

MicroScale Thermophoresis (MST) for studying actin polymerization kinetics.

Here, we present a MicroScale Thermophoresis (MST)-based assay for in vitro assessment of actin polymerization. By monitoring the thermophoretic behavior of ATTO488-labeled actin in a temperature gradient over time, we could follow polymerization in real time and resolve its three characteristic phases: nucleation, elongation, and steady-state equilibration. Titration experiments allowed us to evaluate the effects of actin-binding proteins (ABPs) on polymerization, including DNase I-induced inhibition and mDia2FH1FH2 (mDia2)-assisted acceleration of nucleation. The corresponding rates of actin filament elongation were quantitatively determined, yielding values in good agreement with those obtained using the pyrene-actin polymerization assay. Finally, we measured the effect of myosin on actin polymerization, circumventing the problems of fluorescence quenching and signal disturbance that occur with other techniques. MST is a simple and valuable research tool for investigating actin kinetics covering a wide range of molecular interactions, with low protein consumption.

Assessment of dually labelled PEGylated liposomes transplacental passage and placental penetration using a combination of two ex-vivo human models: the dually perfused placenta and the suspended villous explants.

Uptake and passage of nanocarriers through the placenta are critical information to develop new therapeutic approaches during pregnancy. In order to assess nanocarriers transplacental passage and penetration into the placenta, we studied and optimized two ex-vivo human models: the dually perfused placenta and the placenta explants. Doubly labelled PEGylated liposomes were used as models to provide data on the penetration and transplacental passage of drugs and liposomes. A HPLC method was set-up to quantify both carboxyfluorescein and lipid-rhodamine. Transplacental passage was then quantified using HPLC and placental penetration was assessed using spinning disk microscopy. We found a similar transplacental passage rate for both free and encapsulated carboxyfluorescein as well as a homogeneous fluorescence intensity in the outer cell layer of the placental villous, the syncytiotrophoblast, and the mesenchyma. Besides, liposome-rhodamine was not detected in the fetal circulation. The absence of transplacental passage of PEGylated liposomes is also supported by their detection in the sole syncytiotrophoblast. The combination of two ex-vivo models and the monitoring of both the drug and the carrier provided consistent and complementary information. Overall, we suggest combining the perfused human placenta and the human explants villous models to evaluate nanocarriers designed for treatments during pregnancy.

PDMS-b-PMOXA polymersomes for hepatocyte targeting and assessment of toxicity.

Nanoparticles, such as polymersomes, can be directed to the hepatic asialoglycoprotein receptor to achieve targeted drug delivery. In this study, we prepared asialofetuin conjugated polymersomes based on the amphiphilic di-block copolymer poly(dimethylsiloxane)-b-poly(2-methyloxazoline) (PDMS-b-PMOXA). They had an average diameter of 150nm and formed monodisperse vesicles. Drug encapsulation and sustained release was monitored using the hydrophilic model compound carboxyfluorescein. Asialoglycoprotein receptor specific uptake by HepG2 cells in vitro was energy dependent and could be competitively inhibited by the free targeting ligand. Mechanistic uptake studies revealed intracellular trafficking of asialofetuin conjugated polymersomes from early endosomes and to the lysosomal compartment. Polymersomes showed no toxicity in the MTT assay up to concentrations of 500µg/mL. In addition, acute toxicity and tolerability of our PDMS-b-PMOXA polymersome formulations was assessed in vivo using zebrafish embryos as a vertebrate screening model. In conclusion, a hepatocyte specific drug delivery system was designed, which is safe and biocompatible and which can be used to implement liver-specific targeting strategies.

Assessment of HDACi-Induced Cytotoxicity.

The chromatin contains the genetic and the epigenetic information of a eukaryotic organism. Posttranslational modifications of histones, such as acetylation and methylation, regulate their structure and control gene expression. Histone acetyltransferases (HATs) acetylate lysine residues in histones while histone deacetylases (HDACs) remove this modification. HDAC inhibitors (HDACi) can alter gene expression patterns and induce cytotoxicity in cancer cells. Here we provide an overview of methods to determine the cytotoxic effects of HDACi treatment. Our chapter describes colorimetric methods, like trypan blue exclusion test, crystal violet staining, lactate dehydrogenase assay, MTT and Alamar Blue assays, as well as fluorogenic methods like TUNEL staining and the caspase-3/7 activity assay. Moreover, we summarize flow cytometric analysis of propidium iodide uptake, annexin V staining, cell cycle status, ROS levels, and mitochondrial membrane potential as well as detection of apoptosis by Western blot.

A Nanoparticle-based Sensor Platform for Cell Tracking and Status/Function Assessment.

Nanoparticles are increasingly popular choices for labeling and tracking cells in biomedical applications such as cell therapy. However, all current types of nanoparticles fail to provide real-time, noninvasive monitoring of cell status and functions while often generating false positive signals. Herein, a nanosensor platform to track the real-time expression of specific biomarkers that correlate with cell status and functions is reported. Nanosensors are synthesized by encapsulating various sensor molecules within biodegradable polymeric nanoparticles. Upon intracellular entry, nanosensors reside within the cell cytoplasm, serving as a depot to continuously release sensor molecules for up to 30 days. In the absence of the target biomarkers, the released sensor molecules remain 'Off'. When the biomarker(s) is expressed, a detectable signal is generated (On). As a proof-of-concept, three nanosensor formulations were synthesized to monitor cell viability, secretion of nitric oxide, and ß-actin mRNA expression.

Colorimetric Method of Loop-Mediated Isothermal Amplification with the Pre-Addition of Calcein for Detecting Flavobacterium columnare and its Assessment in Tilapia Farms.

Flavobacterium columnare, the causative agent of columnaris disease in fish, affects many economically important freshwater fish species. A colorimetric method of loop-mediated isothermal amplification with the pre-addition of calcein (LAMP-calcein) was developed and used to detect the presence of F. columnare in farmed tilapia (Nile Tilapia Oreochromis niloticus and red tilapia [Nile Tilapia × Mozambique Tilapia O. mossambicus]) and rearing water. The detection method, based on a change in color from orange to green, could be performed within 45 min at 63°C. The method was highly specific, as it had no cross-detections with 14 other bacterial species, including other fish pathogens and two Flavobacterium species. The method has a minimum detection limit of 2.2 × 10(2) F. columnare CFU; thus, it is about 10 times more sensitive than conventional PCR. With this method, F. columnare was detected in gonad, gill, and blood samples from apparently healthy tilapia broodstock as well as in samples of fertilized eggs, newly hatched fry, and rearing water. The bacteria isolated from the blood were further characterized biochemically and found to be phenotypically identical to F. columnare. The amplified products from the LAMP-calcein method had 97% homology with the DNA sequence of F. columnare.

Rapid cost-effective analysis of microbial activity in soils using modified fluorescein diacetate method.

Fluorescein diacetate (FDA) is commonly used to determine the hydrolyzing activity of microbial organisms in the soil. However, the costs of chemical reagents and time required to perform routine analysis of large number of samples by soil testing laboratories are limiting. Moreover, existing methods generate significant volumes of hazardous waste. In this context, this study was designed to determine the minimum amount of terminating chemical reagent needed to evaluate microbial hydrolyzing activity. The results showed that 0.2 mL of chloroform was enough to effectively stop the hydrolyzing activity in soil. This proposed terminating chemical reagent (0.2 mL chloroform) was also evaluated by comparing with the 10 mL of chloroform and 5 mL of methanol used in the Adam and Duncan method.

The use of fluorescent target arrays for assessment of T cell responses in vivo.

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into > 250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8(+) and CD4(+) T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8(+) T cell-mediated killing of FTA target cells and CD4(+) T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since > 250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g. the cumulative magnitude of the response) and a qualitative (e.g. functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

Rapid aptasensor capable of simply diagnosing prostate cancer.

Using guanine (G)-rich DNA aptamer-conjugated 6-carboxyfluorescein (6-FAM) capable of rapidly capturing prostate specific antigen (PSA) in human serum, cost-effective and simple biosensor with guanine chemiluminescence detection was developed for early diagnosis of prostate cancer. Free G-rich DNA aptamer-conjugated 6-FAM emits bright light in guanine chemiluminescence reaction based on the principle of chemiluminescent resonance energy transfer (CRET). However, G-rich DNA aptamer-conjugated 6-FAM bound with PSA cannot emit light because PSA acts as a strong interference in CRET between 6-FAM and high-energy intermediate formed from the reaction of 3,4,5-trimethoxylphenylglyoxal (TMPG) and guanine of G-rich DNA aptamer. A chemiluminescent biosensor, developed using the different properties of G-rich DNA aptamer-conjugated 6-FAM in the absence and presence of PSA in guanine chemiluminescence reaction, was able to quantify trace levels of PSA in human serum within 30 min without time-consuming and complicated procedures (e.g., multiple incubation and washings) required for conventional immunoassays operated with expensive and intractable antibodies. The limit of detection of chemiluminescent biosensor having a wide linear dynamic range (1.9-125 ng/ml) was 1.0 ng/ml. The excellent correlation (R=0.985) between chemiluminescent biosensor and conventional enzyme immunoassay indicates that the accurate, precise, and rapid chemiluminescent biosensor can be applied as a new method for early diagnosis of prostate cancer.

Development and validation of a LC-MS/MS method for assessment of an anti-inflammatory indolinone derivative by in vitro blood-brain barrier models.

The compound (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-2-one (indolinone) was identified from lipophilic woad extracts (Isatis tinctoria L., Brassicaceae) as a compound possessing potent histamine release inhibitory and anti-inflammatory properties [1]. To further evaluate the potential of indolinone in terms of crossing the blood-brain barrier (BBB), we screened the compound in several in vitro cell-based human and animal BBB models. Therefore, we developed a quantitative LC-MS/MS method for the compound in modified Ringer HEPES buffer (RHB) and validated it according to FDA and EMA guidelines [2,3]. The calibration curve of indolinone in the range between 30.0 and 3000ng/ml was quadratic, and the limit of quantification was 30.0ng/ml. Dilution of samples up to 100-fold did not affect precision and accuracy. The carry-over was within acceptance criteria. Indolinone proved to be stable in RHB for 3h at room temperature (RT), and for three successive freeze/thaw cycles. The processed samples could be stored in the autosampler at 10°C for at least 28h. Moreover, indolinone was stable for at least 16 days in RHB when stored below -65°C. This validation study demonstrates that our method is specific, selective, precise, accurate, and capable to produce reliable results. In the immortalized human BBB mono-culture model, the apparent permeability coefficient from apical to basolateral (PappA→B), and the Papp from basolateral to apical (PappB→A) were 19.2±0.485×10(-6)cm/s and 21.7±0.326×10(-6)cm/s, respectively. For the primary rat/bovine BBB co-culture model a PappA→B of 27.1±1.67×10(-6)cm/s was determined. In the primary rat BBB triple co-culture model, the PappA→B and the PappB→A were 56.2±3.63×10(-6)cm/s and 34.6±1.41×10(-6)cm/s, respectively. The data obtained with the different models showed good correlation and were indicative of a high BBB permeation potential of indolinone confirmed by in silico prediction calculations. P-glycoprotein (P-gp) interaction for indolinone was studied with the aid of a calcein-AM uptake assay, and by calculation of the efflux ratio (ER) from the bidirectional permeability assays. For both bidirectional BBB models an ER below 2 was calculated, indicating that no active mediated transport mechanism is involved for indolinone. In porcine brain capillary endothelial cells (PBCECs), the calcein-AM uptake assay demonstrated that indolinone is neither a P-gp substrate nor a P-gp inhibitor and is accumulated into cells at high extent.

Exclusion-Based Capture and Enumeration of CD4+ T Cells from Whole Blood for Low-Resource Settings.

In developing countries, demand exists for a cost-effective method to evaluate human immunodeficiency virus patients' CD4(+) T-helper cell count. The TH (CD4) cell count is the current marker used to identify when an HIV patient has progressed to acquired immunodeficiency syndrome, which results when the immune system can no longer prevent certain opportunistic infections. A system to perform TH count that obviates the use of costly flow cytometry will enable physicians to more closely follow patients' disease progression and response to therapy in areas where such advanced equipment is unavailable. Our system of two serially-operated immiscible phase exclusion-based cell isolations coupled with a rapid fluorescent readout enables exclusion-based isolation and accurate counting of T-helper cells at lower cost and from a smaller volume of blood than previous methods. TH cell isolation via immiscible filtration assisted by surface tension (IFAST) compares well against the established Dynal T4 Quant Kit and is sensitive at CD4 counts representative of immunocompromised patients (less than 200 TH cells per microliter of blood). Our technique retains use of open, simple-to-operate devices that enable IFAST as a high-throughput, automatable sample preparation method, improving throughput over previous low-resource methods.

Fluorescent labeling of nisin Z and assessment of anti-listerial action.

Biomolecule labeling by fluorescent markers has emerged as an innovative methodology for bio-analytical purposes in food microbiology, medicine and pharmaceutics due to the great advantages of this method such as precision, wide detection limits, and in vivo recognition. Fluorescent nisin Z was synthesized by linking the carboxyl group and amino group of nisin Z and 5-aminoacetamido fluorescein (AAA-flu). This new structure was fully characterized by mass spectrometry with a molecular weight of 3717.3 Da. Intracellular K(+) leakage and transmembrane electrical potential (Δψ) were used to evaluate the antibacterial action of the labeled molecule against three listerial strains and demonstrated that nisin Z endured the labeling process without any activity loss. In vivo activity of labeled nisin was observed by confocal laser microscope which revealed its localization at the septum of listerial cell division site where the membrane-bound cell wall precursor lipid II is maximal. Fluorescent nisin Z showed its great potential as a tool to study antibacterial mechanism of action of nisin in biological systems.

Assessment of the cellular internalization of thermolytic phosphorothioate DNA oligonucleotide prodrugs.

The bioactivity of a CpG-containing phosphorothioate DNA oligonucleotide with thermolytic 2-(N-formyl-N-methylamino)ethyl (fma) thiophosphate groups in mice led us to investigate the parameters affecting the internalization of these thermosensitive DNA prodrugs in various cell lines. Flow cytometry and confocal microscopy analyses indicate that 5'-fluoresceinated fma-phosphorothioate DNA sequences are poorly internalized in Vero, HeLa and GC-2 cells. However, when four fma-thiophosphate groups of a 15-nucleotide long oligothymidylate prodrug are replaced with 3-(N,N-dimethylamino)prop-1-yl thiophosphate functions, internalization of the positively charged prodrug, under physiological conditions, increased fourfold in HeLa and 40-fold in Vero or GC-2 cells. No cytotoxic effects are observed in Vero cells even at an extracellular prodrug concentration of 50 µM over a period of 72 h. Confocal microscopy studies show that internalization of the positively charged oligothymidylate prodrug in Vero cells is time-dependent with early trafficking of the DNA sequence through endosomal vesicles and, eventually, to the nucleus of the cells. Thus, the incorporation of four 3-(N,N-dimethylamino)prop-1-yl thiophosphate groups into thermosentive fma-phosphorothioate DNA prodrugs is an attractive strategy for efficient cellular internalization of these nucleic acid-based drugs for potential therapeutic indications.

Quantitative assessment of cell viability based on flow cytometry and microscopy.

We compare flow cytometric and microscopic determination of cell viability by fluorescence labeling using calcein acetoxy-methyl-ester and ethidium homodimer-1 as live and dead stain, respectively. Peripheral blood monocytes served as model system and were accumulated applying density gradients. Subsequently, monocytes were further enriched by magnetic-activated or fluorescence-activated cell sorting (MACS, FACS) targeting the antigen CD14. Identical samples were used for flow cytometric and microscopic analysis to allow direct comparison of both analysis methods. More than 1,000 cells were measured for each sample to minimize the measurement uncertainty caused by counting statistics. We observed good agreement of flow cytometric and microscopic viability measurements. On average, the difference in viability measured by flow cytometry and microscopy amounted to (2.7 ± 1.4)% for live staining and (1.7 ± 1.2)% for dead staining. These deviations were similar to the uncertainty of measurement for cell viability, thus demonstrating that both methods delivered equal results. Besides monocytes, comparison of flow cytometric and microscopy viability for MACS enriched CD34-positive cells also showed consistent results.

Assessment of fluorescein-based fluorescent dyes for tracing Neotyphodium endophytes in planta.

Fluorescent dyes were assessed for their ability to stain viable hyphae of the fungi Neotyphodium lolii and N. coenophialum, symbiotic endophytes of the Pooideae grasses Lolium perenne and Festuca arundinacea, respectively. The fluorescein-based fluorophores; fluorescein diacetate (FDA), 5(6)-carboxy-fluorescein diacetate (CFDA), 5-chloromethylfluorescein diacetate (CMFDA) and the chitin-binding stain, Calcofluor while M2R, were assessed for staining of endophyte hyphae in vitro from axenic fungal cultures and in planta, including epidermal leaf sheath peels, nodes, ovaries, embryos and meristems. CMFDA produced the greatest intensity of staining of fungal hyphae and gave excellent contrast in planta compared to the plant cells. Compared to the other dyes, CMFDA was also the least affected by photo bleaching and continued to fluoresce up to 2 h after initial excitation. None of the fluorescent dyes stained fungal hyphae in seed.

Detection of hyaluronidase activity using fluorescein labeled hyaluronic acid and Fluorescence Correlation Spectroscopy.

The over-expression of hyaluronidase has been observed in many types of cancer, suggesting that it may have utility for diagnosis. Here we present a technique for the detection of hyaluronidase using Fluorescence Correlation Spectroscopy (FCS). Hyaluronan macromolecules (HAs) have been heavily labeled with fluorescein amine resulting in strong self-quenching. In the presence of hyaluronidase, HA is cleaved into smaller, fluorescein-labeled fragments and the self-quenching is released. Such cleavage is manifested by the increased average diffusion rate of the HA fragments, increased concentration of individual, fluorescent HA fragments, and increased intensity. All three of these properties are monitored simultaneously throughout FCS measurements, both as a function of time and hyaluronidase concentration. The method we present provides a sensitive measure of hyaluronidase activity and requires extremely small amounts of the HA substrate.