DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) for Nucleic Acid Staining
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The Synthesis of Fluorescent 3, 6-Dihydroxyxanthones
University of Wisconsin Milwaukee UWM Digital Commons Theses and Dissertations August 2016 The yS nthesis of Fluorescent 3, 6-dihydroxyxanthones: A Route to Substituted Fluoresceins Surajudeen Omolabake University of Wisconsin-Milwaukee Follow this and additional works at: https://dc.uwm.edu/etd Part of the Chemistry Commons Recommended Citation Omolabake, Surajudeen, "The yS nthesis of Fluorescent 3, 6-dihydroxyxanthones: A Route to Substituted Fluoresceins" (2016). Theses and Dissertations. 1299. https://dc.uwm.edu/etd/1299 This Thesis is brought to you for free and open access by UWM Digital Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UWM Digital Commons. For more information, please contact [email protected]. THE SYNTHESIS OF FLUORESCENT 3, 6-DIHYDROXYXANTHONES: A ROUTE TO SUBSTITUTED FLUORESCEINS by Surajudeen Omolabake A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science In Chemistry at University of Wisconsin-Milwaukee August 2016 ABSTRACT THE SYNTHESIS OF FLUORESCENT 3, 6-DIHYDROXYXANTHONES: A ROUTE TO SUBSTITUTED FLUORESCEIN by Surajudeen Omolabake University of Wisconsin-Milwaukee, 2016 Under the Supervision of Professor Alan W Schwabacher Xanthones belong to the family of compounds of the dibenzo-γ-pyrone framework. Naturally occurring xanthones have been reported to show a wide range of biological and medicinal activities including antifungal,19 antimalarial,20 antimicrobial,21 antiparasitic,22 anticancer,23 and inhibition of HIV activity in cells.24 Xanthones have also been used as a turn on fluorescent probe for metal ions,32 including use as pH indicators, metal ion sensors, in molecular biology, medicinal chemistry and in the construction of other dyes. -
Use of Immunofluorescence and Viability Stains in Quality Control1
Use of Immunofluorescence and Viability Stains in Quality Control1 M. J. CHILVER, J. HARRISON, and T. J. B. WEBB, Process Research Department, Allied Breweries Production Limited, Burton-on- Trent, Staffordshire, England DEM IBZ ABSTRACT has been the failure to differentiate live and dead cells. We have been studying the combined use of immunofluorescent and Immunofluorescence has been evaluated as a rapid quality control viability stains so that live and dead, culture and wild yeasts, can be technique for the detection of low levels of wild yeasts in culture yeast or differentiated on the same slide field. This paper details the results other brewery samples. Antisera have been prepared against antigenic groups A to F, pooled and absorbed with culture yeast. Using the indirect obtained when yeast samples are examined alternately with FITC- staining method and fluorescein isothiocyanate or rhodamine as exciting blue light wavelengths and rhodamine-exciting green light fluorochrome, levels as low as 10 wild yeasts per million cells can be detected and when incident fluorescent light is combined with transmitted in three hours. Combined immunofluorescence and viability staining allows white light to illuminate the specimen. the differentiation of live and dead, culture and wild yeast cells, on the same The potential use of fluorescein diacetate as a viability stain has slide by alternating the light sources. The preferred method uses fluorescein been established. Initial work by Rotman and Papermaster (22) isothiocyanate, excited by incident blue light, for wild yeast detection, was developed by Paton and Jones (21) and its use in brewery combined with methylene blue, viewed by transmitted light, for viability quality control has been described by Molzahn and Portno (19). -
Fluorescein) Injection 10% 2
NEW ZEALAND DATA SHEET 1. PRODUCT NAME FLUORESCITETM (fluorescein) Injection 10% 2. QUALITATIVE AND QUANTITATIVE COMPOSITION FluoresciteTM injection 10% contains 100 mg/mL fluorescein (equivalent to 113.2 mg/mL fluorescein sodium). 3. PHARMACEUTICAL FORM Solution, sterile pyrogen-free injection. Unpreserved with a pH of 8.0 to 9.8. 4. CLINICAL PARTICULARS 4.1. Therapeutic indications Indicated in diagnostic fluorescein angiography or angioscopy of the fundus and of the iris vasculature. 4.2. Dose and method of administration The usual adult dose is the contents of one FluoresciteTM injection 10% vial (5 mL of 10% solution) via intravenous administration. For children, the dose is calculated on the basis of 8 mg/kg of body weight. Parenteral drug products should be inspected visually for particulate matter and discolouration prior to administration. Do not mix or dilute with other solutions or drugs. Flush intravenous cannulae before and after drugs are injected to avoid physical incompatibility reactions. Product is for single use in one patient only. Discard any residue. Contains no antimicrobial agent. Inject the contents of the vial rapidly into the antecubital vein after taking precautions to avoid extravasation. A syringe filled with FluoresciteTM injection 10% is attached to transparent tubing and a 23 gauge butterfly needle for injection. Insert the needle and draw the patient's blood to the hub of the syringe so that a small air bubble separates the patient's blood in the tubing from the FluoresciteTM injection 10%. With the room lights on, slowly inject the blood back into the vein while watching the skin over the needle tip. -
Cell Viability Measurement
Cell Viability Measurement • Viability is a measure of the metabolic state of a cell population which is indicative of the potential for growth • Most common method: membrane integrity – Trypan blue exclusion: leaky membrane allows blue dye to get into cells – Good: capable for manual microscopy – Issues: operator dependent • Cellometer solution: use software algorism – Reduce operator dependency – Reduce calculation error – Speed up counting – Better management of data Nexcelom.com Online Resources [email protected] | 978.327.5340 Cellometer® Simply Counted Trypan Blue Viability for Cell Lines: Exclusion Assay H9 LCL M19 Raw K562 T cell Cellometer has been used for more than 300 cell lines Nexcelom.com Online Resources [email protected] | 978.327.5340 Cellometer® Simply Counted Fluorescence Based Cell Viability Methods • Membrane integrity – DNA binding dye with permeability dependency – AO (acridine Orange): permeable for live and dead cells – PI (propidium iodide), EB (Ethidium bromide), 7-AAD: not permeable for live cells – Others: SYTO ® 13, SYTO 24 ®, SYTO® 14 • Enzymatic activity – Non fluorescent esterases diffuse into cells – Live cells with strong enzymatic activity produce fluorescent products – Live cells retain fluorescent products – Examples: • Calcein-AM to calcein • Fluorescein diacetate (FDA)to fluorescein • Simultaneous detection of both live and dead cell populations for cell lines – Combination of the above with duo-fluorescence detection for each cell sample Nexcelom.com Online Resources [email protected] | 978.327.5340 Cellometer® Simply Counted References [1] S. A. Altman, L. Randers, and G. Rao, "Comparison of Trypan Blue-Dye Exclusion and Fluorometric Assays for Mammalian-Cell Viability Determinations," Biotechnology Progress, vol. 9, pp. 671-674, Nov-Dec 1993. -
Analysis of Dye Mixtures Thin-Layer Chromatography SCIENTIFIC
Analysis of Dye Mixtures Thin-Layer Chromatography SCIENTIFIC Introduction Thin-layer chromatography (TLC) is a valuable analytical technique for separating and analyzing organic compounds. In dye analysis, for example, TLC is used to identify dyes in fabrics or fibers (forensics) and to determine the composition of natural dyes in plants and animals. In this demonstration, thin layer chromatography will be used to separate mixtures of dyes. Concepts • Chromatography • Physical properties Materials Authentic dye samples (eosin Y, fluorescein, methylene blue, and safranin), 0.25 g each Chromatography solvent (acetone), 80 mL Thin layer chromatography sheet (silica gel on plastic backing), 20 cm × 20 cm Beakers, 100-mL, 4 Graduated cylinder, 50-mL Beakers, 400-mL, 4 Metric ruler Beral type pipets, 4 Pencil Microtip pipets, 16 Reaction plate, 24-well Ethyl alcohol, 50 mL Scissors Filter paper Watch glasses, large, or Petri dish covers Safety Precautions Acetone and ethyl alcohol are flammable organic solvents and a dangerous fire risk—keep away from flames, sparks, and other sources of ignition. Work with acetone in a well-ventilated lab only. Wear chemical splash goggles, chemical-resistant gloves, and chemical- resistant apron. Please review current Material Safety Data Sheets for additional safety, handling, and disposal information. Preparation 1. Prepare 0.5% solutions of each dye—dissolve 0.25 g of the dye in 50 mL of water 5 cm (fluorescein, methylene blue, and safranin) or ethyl alcohol (eosin Y). 2. Add about 1 mL of each dye solution to separate wells in a 24-well reaction plate. In other wells, combine 0.5 mL of two or more dyes to prepare dye mixtures. -
Application of Fluorescein Combined with Methylene Blue in Sentinel
www.nature.com/scientificreports OPEN Application of fuorescein combined with methylene blue in sentinel lymph node biopsy of breast cancer Liang Li1, Ning Gao1*, Ai Qing Yang2, Wen Hao Xu1, Yu Ding1, Jun Chu1, Xiao Na Lin1 & Jia Qi Liu1 Sentinel lymph node biopsy (SLNB) for axillary lymph node staging in early breast cancer has been widely recognized. The combination of radio-colloids and dye method is the best method recognized. The reagents and equipment required in the process of the combined method are complex and expensive, so there are certain restrictions in the use of primary medical institutions. As a new tracer, fuorescent tracer technology has attracted much attention. We aimed to evaluate the feasibility and safety of fuorescein for SLNB in breast cancer. In this study, a total of 123 patients with breast cancer were divided into group A (n = 67) and group B (n = 56). The efcacy of Indocyanine green (ICG) combined with methylene blue (group A) and fuorescein combined with methylene blue (group B) in SLNB of breast cancer was compared, complications were observed at the same time. No local or systemic reactions were observed in the two groups. In group A, Sentinel lymph nodes of breast cancer were detected in 63 patients, with a detection rate of 94.0% (63/67), a false-negative rate of 7.5% (4/53). In group B, Sentinel lymph nodes of breast cancer were detected in 52 patients, with a detection rate of 92.9% (52/56), a false-negative rate of 7.5% (3/40). There was no signifcant diference in biopsy results between the two groups. -
Automated Tissue Culture Cell Fixation and Staining in Microplates
Application Note Cell Imaging Automated Tissue Culture Cell Fixation and Staining in Microplates Using the EL406™ Combination Washer Dispenser to Prepare Samples for Imaging with the Cytation™3 Cell Imaging Multi-Mode Reader Paul Held Ph. D., Bridget Bishop, and Peter Banks, Ph. D., Applications Department, BioTek Instruments, Inc., Winooski, VT Fluorescence microscopy has traditionally been performed on microscope slides, but there is a growing trend towards the use of 96- and 384-well microplates as this allows greater number of samples to be easily processed and automated. This is certainly true in the field of High Content Screening. Here we describe the use of the EL406™ Combination Washer Dispenser to automate the fixation and staining processes typically used prior to fluorescence imaging. Introduction The hallmark of fluorescent microscopy is the Phalloidin binds to actin at the junction between use of specific mouse monoclonal antibodies subunits; and because this is not a site at which to recognize and bind to cellular structures. many actin-binding proteins bind, most of the The antibody is a marvelous tool of remarkable F-actin in cells is available for phalloidin labeling [1]. selectivity. It can be used to identify and quantify Because fluorescent phalloidin conjugates are not almost any protein in complex biological matrices. permeant to most live cells, like antibodies, It’s use as a fluorescent marker in microscopy dates they require that cells be either fixed and/or back to the pioneering work of Albert Coons permeablized. Labeled phalloidins have similar directly after World War II, where with colleagues, affinity for both large and small filaments and bind he labeled antipneumococcus antibodies with in a stoichiometric ratio of about one phalloidin anthracene isocyanate and thereby made an molecule per actin subunit. -
Filters for Fluorescence
Optical Filters: Filters for Fluorescence Turan Erdogan, PhD (CTO and Co-founder) Semrock, A Unit of IDEX Corporation May 31, 2011 www.semrock.com Basics of fluorescence • Fluorescence is the property of some atoms and molecules to absorb light over a particular wavelength range and to subsequently emit longer-wavelength light after a brief interval termed the “fluorescence lifetime” . The process of phosphorescence occurs in a manner similar to fluorescence, but with a much longer excited-state lifetime Diagram courtesy of Molecular Expressions™ 2 Fluorescence spectra • In real materials, the vibrational energy levels are not distinguishable, leading to broad smooth spectra • For most common fluorophores, the vibrational energy level spacing is similar for the ground and excited states, resulting in a fluorescence spectrum that strongly resembles the mirror image of the absorption spectrum (i.e., the same transitions are most favorable for both absorption and emission) 3 Fluorescence filters • The purpose of fluorescence filters is to isolate and separate the excitation and emission light • Filters make it possible for the sample to "see" only light within the absorption band, and the detector to "see" only light within the emission band . Without filters, the detector would not be able to distinguish the desired fluorescence from scattered excitation light (especially within the emission band) and autofluorescence from the sample, substrate, and other optics in the system. 4 Fluorescence microscopy Source: Molecular ExpressionsTM Source: -
Basics of Advanced Flow Cytometry
Basics of advanced FlowCytometry Florian Weisel, PhD Laboratory of Mark J. Shlomchik, M.D., Ph.D. Department of Immunology University of Pittsburgh School of Medicine Biomedical Science Tower Pittsburgh, PA FlowCytometry October 1st 2018 email: [email protected] slide 1 Tissue disruption for single-cell suspensions In media or staining buffer vs. Fisher Scientific Part no 1255310 Tissue disruption for single-cell suspensions In media or Staining buffer Tissue disruption for single-cell suspensions 1 2 3 Tissue disruption for single-cell suspensions 4 6 5 7 Organ dissociation gentleMACS Octo Dissociator (Miltenyi) HBSS Mg+ Ca+ ColD (45U/ml) DNaseI (80U/ml) 3% FCS (HI) Heat inactivate FCS: 30min @ 56°C staining Staining Buffer: PBS 3% FCS 0.02 – 0.05% NaN3 (membrane turn-over; contaminations) 2mM EDTA (chelatinG aGent; complexes Ca2+, MG2+) Always stain on ice! Check antibodies for precipitation! Do not use NaN3 if cells are used for functional assays! Stain 6x106 lymphocytes in 50µl (=120x106/ml) For lymphocytes: Spin 14000 rpm (= 370g) 1min per 2ml Cell filtration Elko Filtering co - Nylon Mesh 03-80/37 5 yards $255.1 Enrichment of cell populations examples NEGATIVE SELECTION IS BETTER!!!! …. don’t touch cells of interest Positive / Negative enrichment Cartoon how this works For example: aCD19 – 1mg for $95 Biotinylate in own lab Un-touched enrichment of cell populations A cheaper way Cells + biotinylated ab-cocktail + 80µl 5ml = $790 EasySep 1x109 cells $550 per 65 spleens Miltenyi 1x109 cells $550 1x108 cells 6.5x109 cells Beads are six times cheaper than kit Un-touched enrichment of cell populations Always Do a purity check – just to be sure …. -
DAPI: a DNA-Specific Fluorescent Probe
DAPI: a DNA-Specific Fluorescent Probe Jan Kapuscinski Department of Molecular Biology, Gdansk University, 80-822 Gdansk, Poland, and The Cancer Research Institute, New York Medical College, Elmsford, New York 7 0523 ABSTRACT. DAPI (4’.6-diamidino-2-phenylin- RNase. These observations established DAPI as dole) is a DNA-specific probe which forms a a DNA-specific fluorescent stain. At the same fluorescent complex by attaching in the minor time, these authors discredited DAPI as a quan- grove of A-T rich sequences of DNA It also forms titative measure of DNA because of a mistaken nonflnorescentintercalative complexes with dou- observation that the free dye solution is nearly bk-stranded nucleic acids. The physicochedcal as fluorescent as the DNA-dye complex. Most of the dye and complexes with nu- properties its likely this phenomenon was a result of the pres- cleic acids and history of the development of this dye as a biological stain are described. The appU- ence of sarcosyl (N-laurosylsarcosine), an an- cation of DAPI as a DNA-specMc probe for flow ionic detergent in the solution used for DNA cytometry, chromosome stainfng. DNA visnaliza- separation. Later it was shown that anionic de- tion and qnantitation in histochemistry and bio- tergents enhanced DAPI fluorescence almost as chemistry is reviewed. The mechanisms of DAPI- much as DNA, i.e., about 20 times (Kapuscinski nncleic acid complex formation including minor and Skoczylas 1978). Russell et al. (1975) pub- gmove bding. intercalation and condensation lished a simple cytochemical technique for de- cuediscoseed. tecting myoplasma infection of cultured HeLa cells treated with DAPI using fluorescence mi- Key words: 4’,6-diamidino-2-phenylindolenu- croscopy. -
Enumerating Nucleoid-Visible Marine Bacterioplankton: Bacterial Abundance Determined After Storage of Formalin Fixed Samples Agrees with Isopropanol Rinsing Method
AQUATIC MICROBIAL ECOLOGY Published February 13 Aquat Microb Ecol Enumerating nucleoid-visible marine bacterioplankton: bacterial abundance determined after storage of formalin fixed samples agrees with isopropanol rinsing method Jan H. Vosjan*, Govert J. van Noort Department of Biological Oceanography, Netherlands Institute for Sea Research (NIOZ), PO Box 59,1790 AB Den Burg. Texel, The Netherlands ABSTRACT: The effect of storage of formalin-fixed sea water samples on bacterial abundance deter- mined by DAPI staining and epifluorescence microscopy was compared with the abundance of nucleoid-visible bacteria using isopropanol rinsing after DAPI staining to remove non-specific staining of DAPI. While storage of formalin-fixed water samples at 4'C for 7 mo caused total bacterial numbers to decline exponentially until 50 to 70 d, after which they remained stable, storage of sea water had no effect on the number of nucleoid-visible bacteria over a 7 mo period. Extrapolating the bacterial growth curve of nutrient amended sea water cultures by regression analysis to to resulted in a number of active bacterial cells similar to that obtained by isopropanol rinsing of DAPI-stained samples. This rmght indi- cate that the lag phase commonly detected in sea water cultures is caused by the dormnance of inactive bacteria at the time of inoculation. An intensive field study in the Dutch Wadden Sea showed that the number of nucleoid-visible bacteria continuously declined from around 100% of the total bactenal abundance at the end of the phytoplankton bloom to 75 % within 2.5 mo. Again a strong correlation was detected between nucleoid-visible bacteria determined by DAPI staining and subsequent isopropanol rinsing and total bacterial abundance detectable in the formalin-fixed sea water samples after 7 mo of storage. -
Developing Fluorogenic Reagents for Detecting and Enhancing Bloody Fingerprints
The author(s) shown below used Federal funds provided by the U.S. Department of Justice and prepared the following final report: Document Title: Developing Fluorogenic Reagents for Detecting and Enhancing Bloody Fingerprints Author: Robert M. Strongin, Dr.Martha Sibrian-Vazquez Document No.: 227841 Date Received: August 2009 Award Number: 2007-DN-BX-K171 This report has not been published by the U.S. Department of Justice. To provide better customer service, NCJRS has made this Federally- funded grant final report available electronically in addition to traditional paper copies. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Developing Fluorogenic Reagents for Detecting and Enhancing Bloody Fingerprints Award 2007-DN-BX-K171 Authors Prof. Robert M. Strongin Dr.Martha Sibrian-Vazquez 1 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Abstract Fingerprints are the most common and useful physical evidence for the apprehension and conviction of crime perpetrators. Fluorogenic reagents for detecting and enhancing fingerprints in blood, however, have several associated challenges. For instance, they are generally unsuitable for dark and multi-colored substrates. Luminol and fluorescin and other chemilumigens and fluorigens can be used with dark and often multi-colored substrates, but are not compatible with fixatives and their oxidation products are not insoluble.