DNA Dye Research Articles

Introduction of macarpine as a novel cell‐permeant DNA dye for live cell imaging and flow cytometry sorting

Macarpine (MA) is a quaternary benzophenanthridine plant alkaloid isolated from Macleaya microcarpa or Stylophorum lasiocarpum. Benzophenanthridine alkaloids are interesting natural products that display antiproliferative, antimicrobial, antifungal and anti-inflammatory activities, and also fluorescence properties. In a previous study, we demonstrated that thanks to its ability to interact with DNA and its spectral properties MA could be used as a supravital DNA probe for fluorescence microscopy and flow cytometry including analyses of the cell cycle. In this study, we evaluated the suitability of MA as a DNA dye for time-lapse microscopy and flow-cytometric cell sorting. Living A-375 and MEF cells stained with MA were monitored by time-lapse microscopy for 24 h. Mitoses were observed at MA concentrations up to 0.5 μg/ml during the first 2-3 h. After this period of time, cells treated with MA at concentrations of 0.75 and 0.5 μg/ml underwent apoptosis. Cells cultivated with MA at concentration of 0.25 μg/ml or lower survived throughout the 24 h period. Toxicity of MA was dependent on light wavelength and frequency of image capturing. The intensity of MA fluorescence decreased during the incubation. MA concentration of 0.1 μg/ml was identified as the most suitable for live cell imaging with respect to fluorescence intensity and toxicity. MA at the concentration 10 μg/ml was used for sorting of enhanced green fluorescent protein (EGFP)-labelled neurons and fibroblasts yielding profiles similar to those obtained with DRAQ5. Contrary to DRAQ5, MA-stained cells survived in culture, and the sorted cells lost the MA signal suggesting reversible binding of the dye to the DNA. The results proved that MA may readily be used for chromosomes depicting and mitosis monitoring by time-lapse microscopy. In addition, MA has shown to be a suitable probe for sorting of EGFP-labelled cells, including neurons, that survived the labelling process. In consideration of the results, we highly anticipate an onward use of MA in a broad range of applications based on live cell sorting and imaging, for example, cell synchronisation and monitoring of proliferation as an important experimental and/or diagnostic utility.

Selection of fluorescent DNA dyes for real-time LAMP with portable and simple optics

Loop-mediated isothermal amplification (LAMP) is increasingly used for point-of-care nucleic acid based diagnostics. LAMP can be monitored in real-time by measuring the increase in fluorescence of DNA binding dyes. However, there is little information comparing the effect of various fluorescent dyes on signal to noise ratio (SNR) or threshold time (Tt). This information is critical for implementation with field deployable diagnostic tools that require small, low power consumption, robust, and inexpensive optical components with reagent saving low volume reactions. In this study, SNR and Tt during real-time LAMP was evaluated with eleven fluorescent dyes. Of all dyes tested, SYTO-82, SYTO-84, and SYTOX Orange resulted in the shortest Tt, and SYTO-81 had the widest range of working concentrations. The optimized protocol detected 10 genome copies of Mycobacterium tuberculosis in less than 10min, 10 copies of Giardia intestinalis in ~20min, and 10 copies of Staphylococcus aureus or Salmonella enterica in less than 15min. Results demonstrate that reaction efficiency depends on both dye type and concentration and the selected polymerase. The optimized protocol was evaluated in the Gene-Z™ device, a hand-held battery operated platform characterized via simple and low cost optics, and a multiple assay microfluidic chip with micron volume reaction wells. Compared to the more conventional intercalating dye (SYBR Green), reliable amplification was only observed in the Gene-Z™ when using higher concentrations of SYTO-81.

Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay.

BackgroundThe emergence of Plasmodium falciparum resistance to artemisinins threatens to undermine the effectiveness of artemisinin-based combination anti-malarial therapy. Developing suitable drugs to replace artemisinins requires the identification of new compounds that display rapid parasite killing kinetics. However, no current methods fully meet the requirements to screen large compound libraries for candidates with such properties. This study describes the development and validation of an in vitro parasite viability fast assay for identifying rapidly parasiticidal anti-malarial drugs.MethodsParasite killing kinetics were determined by first culturing unlabelled erythrocytes with P. falciparum in the presence of anti-malarial drugs for 24 or 48 h. After removing the drug, samples were added to erythrocytes pre-labelled with intracellular dye to allow their subsequent identification. The ability of viable parasites to re-establish infection in labelled erythrocytes could then be detected by two-colour flow cytometry after tagging of parasite DNA. Thus, double-stained erythrocytes (with the pre-labelled intracellular dye and the parasite DNA dye) result only after establishment of new infections by surviving parasites. The capacity of the test anti-malarial drugs to eliminate viable parasites within 24 or 48 h could, therefore, be determined.ResultsThe parasite viability fast assay could be completed within 48 h following drug treatment and distinguished between rapidly parasiticidal anti-malarial drugs versus those acting more slowly. The assay was validated against ten standard anti-malarial agents with known properties and results correlated well with established methods. An abbreviated assay, suitable for adaption to medium–high throughput screening, was validated and applied against a set of 20 compounds retrieved from the publically available Medicines for Malaria Venture ‘Malaria Box’.ConclusionThe quantification of new infections to determine parasite viability offers important advantages over existing methods, and is amenable to medium–high throughput screening. In particular, the parasite viability fast assay allows discrimination of rapidly parasiticidal anti-malarial candidates.

CTAB enhancement of FRET in DNA structures.

The effect of cetyl-trimethylammonium bromide (CTAB) on enhancing the fluorescence resonance energy transfer (FRET) between two dye-conjugated DNA strands was studied using fluorescence emission spectroscopy and dynamic light scattering (DLS). For hybridized DNA where one strand is conjugated with a TAMRA donor and the other with a TexasRed acceptor, increasing the concentration of CTAB changes the fluorescence emission properties and improves the FRET transfer efficiency through changes in the polarity of the solvent, neutralization of the DNA backbone and micelle formation. For the DNA FRET system without CTAB, the DNA hybridization leads to contact quenching between TAMRA donor and TexasRed acceptor producing reduced donor emission and only a small increase in acceptor emission. At 50 µM CTAB, however, the sheathing and neutralization of the dye-conjugated dsDNA structure significantly reduces quenching by DNA bases and dye interactions, producing a large increase in FRET efficiency, which is almost four fold higher than without CTAB.

Localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution

Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant® DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei of fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 106 signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy.

Abstract 202: Four dimensional quantitative label-free holographic imaging of the cell cycle in tumor cell lines

Abstract Introduction: Quantitative analytical systems have the ability to quantify the amount of constituents on a per entity basis. For example, flow cytometers use stoichiometric DNA stains to quantify the amount of DNA on a per cell basis, and allow discrimination of the cell cycle phases G0/G1, S-Phase, and G2/M. Quantitative imaging cytometers employ morphometric features (total DNA content, DNA maximum brightness, and nuclear area) to track the cell cycle during the mitotic phases. As cells enter mitosis, the condensation of the chromatin to form chromosomes increases the DNA density, while the nuclear area and volume decrease. Unfortunately, DNA dyes are harmful for live cells and can interfere with the results of toxicological assays. It is desirable to have a method for label-free long-term evaluation of the cell cycle in living cells. Methods: The Holomonitor®M4 (Phase Holographic Imaging, Lund, Sweden) is an incubator-adapted time-lapse imaging system that records interference patterns from a low power 635nm diode laser. Computer software is used to de-convolve the interference pattern at a plurality of heights to form 3-dimensional and 2-dimensional images of the sample thickness. These images are segmented and features (cellular area, optical thickness and volume, etc.) are calculated on a per cell basis. Positional information is recorded for each cell. Data can be presented as positional track charts, position vs. feature graphs, and feature vs. feature graphs. We obtained time-lapse videos using proprietary PHI software and also generated 4-dimensional plots (X-position, Y- position, optical thickness coded as the pixel intensity, and time) in ImageJ software. Results: In our studies of SKOV3 tumor cell lines, we found a set of cellular based features (cell volume, optical thickness, and cell area) that correlate with the DNA-based features we are accustomed to using in our traditional label-based systems. In the 4D plots, the history of each cell within the imaging area is presented for the entire evaluation time - typically 18-24 hours with 5 minute sampling increments. With a low optical thickness threshold, tracks of all of the cells are displayed. The image intensity threshold can be raised, so only mitotic events are displayed. Mitotic cells have a characteristic Y shaped tracks, appearing suddenly as the chromatin condenses, bifurcating as the daughter cells move away from each other, and then disappearing as the chromatin de-condenses. Cells blocked in mitosis present as persistent long tracks. The rapid degradation of apoptotic cells is often visible, while cells undergoing mitotic catastrophe suddenly disappear. Conclusions: Time-lapse label-free holographic imaging combining traditional analysis and novel display techniques is well suited for toxicological studies and visualization of drug effects on the cell cycle. Citation Format: Ed Luther, Jeffrey Agar, Mansoor Amiji. Four dimensional quantitative label-free holographic imaging of the cell cycle in tumor cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 202. doi:10.1158/1538-7445.AM2015-202

Abstract 4923: Detecting copy number variation using dNTP limited PCR and high-resolution melting

Abstract Background: Copy number variation (CNV) is a common type of genetic variation. About 13% of genes in the human genome have variation in copy number. Copy number alterations (CNAs), somatic changes to chromosome structure that result in gain or loss in copies of sections of DNA, are very common in cancer and associated with particular cancer types. In some cases such as EGFR and HER2, the copy number alterations have led to the identification of cancer-causing genes and suggested specific therapeutic approaches. Materials and Methods: Target and reference regions of genomic DNA were amplified together using a variety of amplification limiting PCR reagent and cycling protocols. Ratios of reference and copy number variants were maintained by limiting quantities of dNTPs and allowing PCR to plateau. Suitable fragments having single melting domains, well-separated Tms, and no common homologs were designed using uMelt melting curve prediction software. After PCR, high-resolution melting (fluorescence vs. temperature) data was acquired from double stranded DNA dye LCGreen Plus present in the reaction. MeltWizard 6 software was used to remove background, equalize negative derivative melting peaks corresponding to the reference segment, and identify and quantify samples with different CNV ratios from the amplitude of their target melting peaks. The protocols were optimized on samples with known ratios. Results: All proposed methods of limiting amplification (cycle number, dNTPs, Taq polymerase) were found to be capable of preserving initial copy ratios sufficiently to identify all common human copy number variation ratios when used in conjunction with high-resolution melting peak reference normalization. CNV ratios far beyond this range, as small as 2 : 2.125 (5.88%), were detectable using the most robust, simplest, and highest resolution method of limiting dNTPs at an optimal 3.25 uM. Duplex PCR of reference CFTR and target EGFR genes were used to detect the copy number variation. Copy number of EGFR in 8 NSCNC lung cancer and 41 colon cancer samples were detected with 6.25 uM dNTP. In 7 out of 8 of lung cancer and 7 out of 41 colon cancer samples, the copy number of EGFR is higher than normal copy 2. The Tm of the reference peak may be lower (duplication) or higher (deletion) than that of the CNV target without affecting the results. Conclusion: Copy number variation can be quantified using reaction limited multiplex PCR followed by high-resolution melting analysis. This simple closed-tube method is fast, economical, more accurate and less susceptible to contamination than other methods. Assays are easy to design, limiting dNTPs is simple, and the results are stable and reliable. After PCR, only 5 to 10 minutes of melting and analysis are required to obtain results. Citation Format: Luming Zhou, Bobert Palais, Carl Wittwer. Detecting copy number variation using dNTP limited PCR and high-resolution melting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4923. doi:10.1158/1538-7445.AM2015-4923

Abstract 2703: Overcoming Lapatinib resistance by the fatty acid synthase inhibitor HS-106

Abstract Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that is overexpressed in about 20% of breast cancers and most often in the more aggressive inflammatory breast cancer (IBC). Lapatinib is one of the most potent small molecule inhibitors of HER2. Although Lapatinib has shown to be an effective targeted therapy, development of drug resistance stands as a major problem. Here we show that the anti-cancer activity of the novel fatty acid synthase inhibitor HS-106 can re-sensitize resistant cell lines to Lapatinib. Lapatinib resistant SKBR3 and BT474 breast cancer cell lines were treated with increasing concentrations of HS-106 either alone or in combination with Lapatinib and their effects on cell proliferation were assessed using the DNA dye Hoechst 33342. The ability of HS-106 and Lapatinib drug combination to induce apoptosis were measured in Caspase 3/7 activity assay using the fluorogenic Caspase substrate (DEVD)2 R110. Their effect on apoptosis was further confirmed in Lapatinib resistant BT474 cells using flow cytometry based Annexin V assay. In addition, the effect of HS-106 on the expression levels and phosphorylation of the kinases involved in Her2 pathway including Her2, AKT and ERK were determined. The fatty acid synthase inhibitor HS-106 significantly inhibits the proliferation of Lapatinib resistant cell lines with concentrations. Interestingly, when combined with Lapatinib, the drugs synergize to dramatically affect cell growth. This anti-proliferative activity of HS-106 was found to be the result of induction of apoptosis, Caspase 3/7 activity was found to increase in a dose dependent manner following HS-106 treatment. Low concentrations of Lapatinib and HS-106, which does not induce Caspase activity when administrated separately, were found to significantly induce Caspase activity when combined together. These results were confirmed with the Annexin V assay. By examining the expression levels of HER2 pathway proteins we found a decrease in HER2 levels which was reflected on the levels of the downstream kinases AKT and ERK when treating with high concentrations of HS-106. In a previous work, HS-106 showed a good tolerability and anti-cancer activity in the MMTV neu+ mouse model. By showing potent efficacy against Lapatinib resistant breast cancer cell lines, our data suggest that HS-106 is an excellent drug candidate to advance into translational studies for the treatment of Lapatinib resistant breast cancer. Citation Format: Yazan Alwarawrah, Philip Hughes, Rachid Safi, Donald P. McDonnell, Neil L. Spector, Timothy A. Haystead. Overcoming Lapatinib resistance by the fatty acid synthase inhibitor HS-106. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2703. doi:10.1158/1538-7445.AM2015-2703

Upgrading the GSI beamline microscope with a confocal fluorescence lifetime scanner to monitor charged particle induced chromatin decondensation in living cells

We report the upgrade of the GSI beamline microscope coupled to the linear accelerator UNILAC by a confocal FLIM scanner utilizing time correlated single photon counting technique (TCSPC). The system can now be used to address the radiation induced chromatin decondensation in more detail and with higher sensitivity compared to intensity based methods. This decondensation of heterochromatic areas is one of the early DNA damage responses observed after charged particle irradiation and might facilitate the further processing of the induced lesions. We describe here the establishment of different DNA dyes as chromatin compaction probes usable for quantification of the DNA condensation status in living cells utilizing lifetime imaging. In addition, we find an evidence of heterochromatic chromatin decondensation in ion irradiated murine chromocenters detected after subsequent fixation using FLIM measurements.

An assay to probe Plasmodium falciparum growth, transmission stage formation and early gametocyte development.

Conversion from asexual proliferation to sexual differentiation initiates the production of the gametocyte, which is the malaria parasite stage required for human-to-mosquito transmission. This protocol describes an assay designed to probe the effect of drugs or other perturbations on asexual replication, sexual conversion and early gametocyte development in the major human malaria parasite Plasmodium falciparum. Synchronized asexually replicating parasites are induced for gametocyte production by the addition of conditioned medium, and they are then exposed to the treatment of interest during sexual commitment or at any subsequent stage of early gametocyte development. Flow cytometry is used to measure asexual proliferation and gametocyte production via DNA dye staining and the gametocyte-specific expression of a fluorescent protein, respectively. This screening approach may be used to identify and evaluate potential transmission-blocking compounds and to further investigate the mechanism of sexual conversion in malaria parasites. The full protocol can be completed in 11 d.

In vivo assessment of rodent Plasmodium parasitemia and merozoite invasion by flow cytometry.

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

Cell cycle staging of individual cells by fluorescence microscopy.

Progression through the cell cycle is one of the most fundamental features of cells. Studies of the cell cycle have traditionally relied on the analysis of populations, and they often require specific markers or the use of genetically modified systems, making it difficult to determine the cell cycle stage of individual, unperturbed cells. We describe a protocol, suitable for use in high-resolution imaging approaches, for determining cell cycle staging of individual cells by measuring their DNA content by fluorescence microscopy. The approach is based on the accurate quantification by image analysis of the integrated nuclear intensity of cells stained with a DNA dye, and it can be used in combination with several histochemical methods. We describe and provide the algorithms for two automated image analysis pipelines and the derivation of cell cycle profiles with both commercial and open-source software. This 1-2-d protocol is applicable to adherent cells, and it is adaptable for use with several DNA dyes.

Potential of PAR1 to be involved in the acquisition of drug resistance in human gastric cancer cell.

94 Background: Side Population (SP) is a FCM term to define cell clusters with strong ability to efflux DNA dye Hoechst 33342 by ABC transporters. In some cases, cancer stem-like cells (CSLCs) were identified using the FCM-based SP technique and SP cells had ability of drug resistance because SP cells were expressed ABC transporter. CSLCs may generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. We had showed PAR1 had an intimate involved in gastric cancer metastasis. And PAR1 contributed to leukemic stem cell maintenance. So we studied PAR1 induced SP cells acquired the ability of drug resistance in gastric cancer. Methods: MKN45 cells stably expressing PAR1 (45-PAR1) were generated in our laboratory. siRNA against PAR1 was synthesized. We studied the change in fraction of SP cell, when PAR1-expressing cell (45-PAR1 and MKN74), PAR1-null expressing cell MKN45 and inhibited PAR1-expressing MKN74 by PAR1 siRNA (74-PAR1siRNA) were treated with PAR1 agonist TFLLR-NH2 or TFLLR-NH2 plus PAR1 selective antagonist SCH79797. And, we compared the drug resistance using paclitaxel, cisplatin and 5-FU and researched ABC transporter expression in MKN45, 45-PAR1 and MKN74 with TFLLR-NH2 or SCH79797 or PAR1 siRNA. Results: We showed the fraction of SP cells in 45-PAR1 and MKN74 achieved dramatic increases by TFLLR-NH2. But, the fraction of SP cells in MKN45 and 74-PAR1siRNA remained about the same by TFLLR-NH2. When treated with SCH79797, the fraction of SP cells remained about the same, too. As for drug resistance assay, 45-PAR1 and MKN74 achieved the same strong resistance of the SP cells by TFLLR-NH2. But, MKN45 and 74-PAR1siRNA were not able to obtain the drug resistance. When treated with TFLLR-NH2, 45-PAR1 and MKN74 showed the high expression of ABCG1 and MRP1, that proteins are ABC transporters, compared to MKN45 and 74-PAR1siRNA. These expression levels in 45-PAR1 and MKN74 with TFLLR-NH2 were equal to the SP cells. Conclusions: PAR1 is closely-involved in that gastric cancer cells achieved the drug resistance and were led to SP enriched in CSLCs.

Facile and Cost-Effective Detection of Saxitoxin Exploiting Aptamer Structural Switching.

A simple method to detect saxitoxin (STX), one of the main components of the paralytic shellfish poison from red tide, has been developed. By using a next generation dye for double-stranded DNA we were able to differentiate fluorescence from STX-binding aptamers when exposed to different concentrations of STX, suggesting a change in aptamer folding upon target binding. The developed method is extremely rapid, only requiring small sample volumes, with quantitative results in the concentration range of 15 ng/mL to 3 µg/mL of STX, with a detection limit of 7.5 ng/mL.

Measuring DNA content by flow cytometry in fission yeast.

Flow cytometry is an essential tool to monitor DNA content and determine cell cycle distribution. Its utility in fission yeast reflects the ease of sample preparation, the stochiometric binding of the most popular DNA dyes (propidium iodide and Sytox Green), and ability to monitor cell size. However, the study of DNA replication with multicolour flow analysis has lagged behind its use in mammalian cells. We present basic and advanced protocols for analysis of DNA replication in fission yeast by flow cytometry including whole cell, nuclear "ghosts," two-color imaging with BrdU, and estimates of DNA synthesis using EdU.

Melting analysis on microbeads in rapid temperature-gradient inside microchannels for single nucleotide polymorphisms detection.

A continuous-flow microchip with a temperature gradient in microchannels was utilized to demonstrate spatial melting analysis on microbeads for clinical Single Nucleotide Polymorphisms (SNPs) genotyping on animal genomic DNA. The chip had embedded heaters and thermometers, which created a rapid and yet stable temperature gradient between 60 °C and 85 °C in a short distance as the detection region. The microbeads, which served as mobile supports carrying the target DNA and fluorescent dye, were transported across the temperature gradient. As the surrounding temperature increased, the fluorescence signals of the microbeads decayed with this relationship being acquired as the melting curve. Fast DNA denaturation, as a result of the improved heat transfer and thermal stability due to scaling, was also confirmed. Further, each individual microbead could potentially bear different sequences and pass through the detection region, one by one, for a series of melting analysis, with multiplex, high-throughput capability being possible. A prototype was tested with target DNA samples in different genotypes (i.e., wild and mutant types) with a SNP location from Landrace sows. The melting temperatures were obtained and compared to the ones using a traditional tube-based approach. The results showed similar levels of SNP discrimination, validating our proposed technique for scanning homozygotes and heterozygotes to distinguish single base changes for disease research, drug development, medical diagnostics, agriculture, and animal production.

Association of neutrophil extracellular traps with endometriosis-related chronic inflammation

ObjectiveTo study if neutrophil extracellular traps (NETs) are present in the peritoneal fluid of endometriosis patients. NETs play a crucial role in fighting against microorganisms. However, exaggerated NET production may lead to tissue damage in their vicinity in pathological conditions. Our study evaluates the presence of NETs in endometriosis peritoneal fluid. Study designPeritoneal fluid (PF) was collected in a case-control study from 52 women, who underwent either diagnostic or operative laparoscopy. The control group consisted of 17 women with infertility, chronic pelvic pain, simple or functional cysts or irregular bleeding. The endometriosis group, altogether 35 patients, comprised 19 patients with stage I and II and 16 patients with stage III and IV endometriosis. First we tested whether the PF is able to stimulate NET production. Neutrophils from healthy volunteers were treated with the PF of endometriosis patients and controls and NETs were detected with Sytox orange extracellular DNA dye and immunofluorescence microscopy. Then we evaluated if NETs were already present in the collected PF using the specific myeloperoxidase (MPO)-DNA capture ELISA method, based on the MPO associated with the NET scaffold. ResultsThe PF of endometriosis patients did not stimulate NET release from healthy granulocytes. However, pre-existent NETs could be detected in 17 endometriosis patients out of 35 (49%). In contrary, in the control group NETs were present in only 3 patients out of 17 (18%), (p=0.03, OR: 4.4). Moreover, the quantification of NETs showed a significantly higher amount of NETs in endometriosis compared to the controls (0.097 vs. 0.02, p=0.04). ConclusionThis is the first study, which evaluated and described the presence of NETs in the PF of endometriosis patients. Our study shows, that NETs may be involved in the complex pathophysiology of endometriosis.

Prognostic value of cell-mediated immunity changes in patients with diabetes mellitus and metabolic syndrome undergoing simultaneous surgical operations

Aim. To study the prognostic value of cell-mediated immunity changes in patients with type II diabetes mellitus undergoing simultaneous operations for treatment of cholelithiasis and postoperative ventral hernias. Methods. Two groups of female patients with cholelithiasis and postoperative ventral hernias who underwent planned simultaneous surgical treatment were examined. The firs group included 48 patients with type II diabetes mellitus and abdominal obesity. The second group included 48 patients without diabetes mellitus and metabolic syndrome. Mononuclear leukocytes of peripheral blood were isolated by density gradient centrifugation on Ficoll-verografin. Relative and absolute number of lymphocyte populations and subpopulations and FAS-receptor expression were studied by indirect immunofluorescence using specific monoclonal antibodies. Apoptotic cells were determined by fluorescent microscopy after exposure of separated mononuclear leukocytes to DNA dye Hoechst 33342. Results. Patients with type II diabetes mellitus had higher incidence of purulent complications after laparoscopic cholecystectomy and surgical treatment of ventral hernias (14.6% vs. 2.0%, p 0.01), which was associated with absolute and relative decrease of CD4+ and CD16+ lymphocyte levels and immunoregulatory index and increased spontaneous and FAS-induced apoptosis of mononuclear leukocytes. In patients who developed purulent complications, more significant reduction of the absolute number of CD16+ lymphocytes (34.5±3.8×106/L vs. 76.72±5.16×106/L, p 0.01) and increased spontaneous apoptosis (7.9%±1.1% vs. 2.7±0.3% compared to patients without complications, p 0.01) was determined. Conclusion. Immunological predictors for developing postoperative purulent complications after simultaneous abdominal surgeries in patients with type II diabetes include decreased indicators of spontaneous apoptosis and decreased number of NK-cells.

Real-time PCR-based genotyping from whole blood using Taq DNA polymerase and a buffer supplemented with 1,2-propanediol and trehalose

Amplification of DNA templates from whole blood with Taq DNA polymerase remains a difficult task worldwide. Using a real-time PCR setup and a buffer supplemented with 1M 1,2-propanediol, 0.2M trehalose, and SYBR green I we show a reliable technique for genotyping in mice and detection of single-nucleotide polymorphisms/mutations in humans. Elimination of DNA extraction and use of the common Taq DNA polymerase and DNA dye bring about substantial savings in labor and cost.

High UV Excitation Intensity Induces Photoconversion of DAPI During Wide-Field Microscopy

Background: Multi-color fluorescence microscopy is dependent on the spectral specificity of the dyes and probes used for localization. One of the most commonly used fluorescent DNA dyes is DAPI, which is usually excited by UV light to emit in the blue visible light range. Herein, we describe a pattern of decreasing DAPI fluorescence upon extended UV exposure, closely followed by an increase in emission maxima in the green range.
 Methods: UV-induced photo-conversion of DAPI to green-fluorescing photoproducts was studied on Chinese hamster ovary cells using wide-field fluorescence microscopy, at different UV exposure times and intensities. Imaging was done in repetitive cycles, by alternating between a DAPI and FITC filter cube and following this with a 1 second UV excitation time. The effect of differing UV light intensities on the photo- conversion process was not previously described in the literature.
 Results: Upon image analysis from a large sample of cells, the rate of photo-conversion was shown to be dependent on both the duration of UV excitation and the intensity of the UV light source. Both the process of DAPI depletion and photo-product growth showed biphasic exponential patterns of change. Furthermore, the level of DAPI fluorescence intensity was found to be negatively correlated with the green fluorescence of the photo-product.
 Limitations: This study did not examine the effect of differing mounting media or a variation in DAPI concentration on the rate of DAPI photo-conversion. Also, the exact light dosage to the system was not measured from the 100W Hg bulb. Photo-bleaching of green fluorescence in cells not stained with DAPI was not measured to control for bleaching of endogenous cell molecules.
 Conclusion: Based on our findings, a set of recommendations was formulated in order to help reduce the effects of UV-induced DAPI photo-conversion.