LIVE/DEAD Viability/Cytotoxicity
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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. -
Fluorescence-Based in Situ Assay to Probe the Viability and Growth
Avalos Vizcarra et al. Biointerphases 2013, 8:22 http://www.biointerphases.com/content/8/1/22 ORIGINAL ARTICLE Open Access Fluorescence-based in situ assay to probe the viability and growth kinetics of surface-adhering and suspended recombinant bacteria Ima Avalos Vizcarra1, Philippe Emge1, Philipp Miermeister2, Mamta Chabria1, Rupert Konradi3, Viola Vogel1 and Jens Möller1* Abstract Bacterial adhesion and biofilm growth can cause severe biomaterial-related infections and failure of medical implants. To assess the antifouling properties of engineered coatings, advanced approaches are needed for in situ monitoring of bacterial viability and growth kinetics as the bacteria colonize a surface. Here, we present an optimized protocol for optical real-time quantification of bacterial viability. To stain living bacteria, we replaced the commonly used fluorescent dye SYTO® 9 with endogenously expressed eGFP, as SYTO® 9 inhibited bacterial growth. With the addition of nontoxic concentrations of propidium iodide (PI) to the culture medium, the fraction of live and dead bacteria could be continuously monitored by fluorescence microscopy as demonstrated here using GFP expressing Escherichia coli as model organism. The viability of bacteria was thereby monitored on untreated and bioactive dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAC)-coated glass substrates over several hours. Pre-adsorption of the antimicrobial surfaces with serum proteins, which mimics typical protein adsorption to biomaterial surfaces upon contact with host body fluids, completely blocked the antimicrobial activity of the DMOAC surfaces as we observed the recovery of bacterial growth. Hence, this optimized eGFP/PI viability assay provides a protocol for unperturbed in situ monitoring of bacterial viability and colonization on engineered biomaterial surfaces with single-bacteria sensitivity under physiologically relevant conditions. -
Assessment of Islet Cell Viability Using Fluorescent Dyes
Diabetologia (1987) 30:812-816 Diabetologia Springer-Verlag 1987 Assessment of islet cell viability using fluorescent dyes H. L. Bank Department of Pathologyand LaboratoryMedicine, Medical Universityof South Carolina, Charleston, South Carolina, USA Summary. A rapid fluorometfic method has been developed of islet cells prior to time-consuming experiments rather than to evaluate the viability of isolated islet cells. The assay dif- retrospectively. This assay can also be used with intact islets. ferentiates between viable and nonviable cells by the simulta- Stained islets can be divided into three distinct groups: green neous use of the inclusion and exclusion dyes acridine fluorescing islets contain insulin, red fluorescing islets con- orange and propidium iodide. When viewed by fluorescent tain little or no insulin and a third class of islets containing microscopy, viable cells fluoresce green, while nonviable some non-viable cells fluoresce red, green, and yellow. The cells fluoresce bright red. Although the acridine orange and yellow colour is due to the superimposition of red and green propidium iodide assay measures membrane integrity, the re- fluorescing cells. sults of this assay correlate with other measures of cell viabil- ity. Compared to trypan blue exclusion, this assay is easier to Key words: Islet viability, fluorochromes, B cells, acridine read, more stable, and has fewer staining artifacts. The assay orange, propidium iodide, trypan blue exclusion, membrane enables the rapid estimation of the viability of a population integrity. A reliable method for rapidly determining the viability rescence. Propidium iodide (PI) is impermeable to in- of isolated islet cells would be useful in studies on tact plasma membranes, but it easily penetrates the transplantation, cryopreservation, insulin release, and plasma membrane of dead or dying cells [7] and inter- biosynthesis. -
3D Cell Culture HTS Cell Viability Complete Assay Kit 09/16 (Catalog #K948-100; 100 Assays; Store at -20°C)
FOR RESEARCH USE ONLY! 3D Cell Culture HTS Cell Viability Complete Assay Kit 09/16 (Catalog #K948-100; 100 assays; Store at -20°C) I. Introduction: Three dimensional (3D) cell cultures are artificially-created environments in which cells are permitted to grow or interact with their surroundings in a 3D fashion. 3D cell cultures improve the function, differentiation and viability of cells and recapitulate in vivo microenvironment compared to conventional 2D cell cultures. 3D matrices provide a physiologically relevant screening platform, by mimicking the in vivo responses, for many cell types including cancer and stem cells in developmental morphogenesis, pharmacology, drug metabolism and drug toxicity studies. Quantification of number of viable cells is an indispensable tool in in vitro screening in these studies. Calcein AM is a non-fluorescent, hydrophobic compound that easily penetrates intact and live cells, and has been widely used to assess cell viability and proliferation in Cell Biology research. However, with the use of 3D matrices, some proteases-based dissociation methods don’t completely dissolve the matrices and cell aggregates, which could alter the result in quantitative in vitro assays such as viability assessment. BioVision’s 3D Cell Culture Cell Viability Complete Assay Kit provides a standardized fluorometric method for sensitive quantification of viable cells that can detect as low as 50 viable cells in each well and can be measured at Ex/Em = 485/530 nm. The measured fluorescence intensity is proportional to the number of viable cells. Further, as a complete set, the kit comes with an optimized and gentle non-enzymatic dissociation solution for the recovery of viable and dead cells from spheroids in matrices and scaffolds. -
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). -
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) for Nucleic Acid Staining
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) for nucleic acid staining Catalog number: AR1176-10 Boster’s DAPI solution is a fluorescent dye with higher efficiency for immunofluorescence microscopy. This package insert must be read in its entirety before using this product. For research use only. Not for use in diagnostic procedures. BOSTER BIOLOGICAL TECHNOLOGY 3942 B Valley Ave, Pleasanton, CA 94566 Phone: 888-466-3604 Fax: 925-215-2184 Email:[email protected] Web: www.bosterbio.com DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) for nucleic acid staining Catalog Number: AR1176-10 Product Overview Material DAPI dihydrochloride (MW = 350.3) Form Liquid Size 10 mL(100 assys) Concentration 1μg/ml 8 mM sodium phosphate, 2 mM potassium phosphate, 140 mM sodium chloride, 10 Buffer mM potassium chloride; pH 7.4. Storage Upon receipt store at -20°C, protect from light. Stability When stored as directed, product should be stable for one year. Molecular formula C16H15N5 • 2 HCL Excitation: DAPI 340nm Emission: DAPI 488nm Excitation: DAPI-DNA complexes 360nm Emission: DAPI-DNA complexes 460nm Thermofisher (Product No. 62247); Thermofisher (Product No. 62248); Millipore Equivalent Sigma (Product No. D9542) BOSTER BIOLOGICAL TECHNOLOGY 3942 B Valley Ave, Pleasanton, CA 94566 Phone: 888-466-3604 Fax: 925-215-2184 Email:[email protected] Web: www.bosterbio.com Notes: Type of DAPI Molecular formula Molecular weight Catalog Number DAPI dihydrochloride C16H15N5 • 2 HCL 350.25 AR1176-10 DAPI dilactate C16H15N5 • 2 C3H6O3 457.48 N/A Introduction DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye which can bind DNA strands robustly, the fluorescence being detected by fluorescence microscope. -
Cell Viability, Proliferation and Cytotoxicity Assays Annexin V Apoptosis Detection Kits
Cell Viability, Proliferation and Cytotoxicity assays Annexin V Apoptosis Detection Kits Description: Includes for 100 assays: Annexin V Apoptosis Detection Kits is a convenient, · 500 μl Labeled Annexin V easy-to-use and safe method for Apoptosis Detection. · 50 mL Binding Buffer (10x) Annexins are a family of calcium-dependent · 500 μl Propidium iodide phospholipid-binding proteins, which bind to phosphatidylserine (PS). Applications: Detect early/middle stages of apoptosis. Externalization of phosphatidylserine residues on Differentiate apoptosis from necrosis. the outer plasma membrane of apoptotic cells allows detection via Annexin V. Once the apoptotic cells are Related Products: bound with labelled Annexin V, it can be visualized · XTT Cell Proliferation Assay Kit (p.78) with fluorescent microscopy or cytometry. Ordering info: Since loss of membrane integrity is a pathognomonic feature of necrotic cell death, necrotic cells will stain Annexin V-FITC with specific membrane-impermeant nucleic acid dyes Cat No. Size such as propidium iodide, the membrane integrity of CA011 100 assays apoptotic cells can be demonstrated by the exclusion Annexin V-APC of these dyes. Cat No. Size CA012 100 assays Advantages & Features: Annexin V-Biotin Easy and fast protocol. Cat No. Size Versatile: proven performance for both adherent CA013 100 assays and suspension cells. Annexin V-PE Safe: non-enzymatic assay that avoids fixation. Cat No. Size CA014 100 assays XTT Cell Proliferation Assay Kit Description: Applications: XTT Cell Proliferation Assay Kit is an optimized, Spectrophotometric quantification of cell accurate and sensitive colorimetric assay that detects proliferation and viability in response to the cellular metabolic activities. During the assay, pharmaceutical, chemical, nutrients and the yellow tetrazolium salt XTT (sodium 2,3,-bis(2- environmental compounds. -
Induction of Apoptosis of Cancer Cells Using the Cisplatin Delivery Based Electrospray (CDES) System
applied sciences Communication Induction of Apoptosis of Cancer Cells Using the Cisplatin Delivery Based Electrospray (CDES) System Myung Chul Lee 1,† , Shambhavi Pandey 2,†, Jae Woon Lim 3,†, Sangbae Park 3 , Jae Eun Kim 4, Hyunmok Son 3, Jinsub Han 4,5,6, Hoon Seonwoo 7,8,*, Pankaj Garg 2,* and Jong Hoon Chung 2,4,5,6,* 1 Division of Engineering in Medicine Brigham and Women’s Hospital Department Harvard Medical School, Cambridge, MA 02139, USA; [email protected] 2 Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea; [email protected] 3 Department of Biosystems & Biomaterial Science and Engineering, Seoul National University, Seoul 08826, Korea; [email protected] (J.W.L.); [email protected] (S.P.); [email protected] (H.S.) 4 Department of Biosystems Engineering, Seoul National University, Seoul 08826, Korea; [email protected] (J.E.K.); [email protected] (J.H.) 5 BK21 Global Smart Farm Educational Research Center, Seoul National University, Seoul 08826, Korea 6 Convergence Major in Global Smart Farm, Seoul National University, Seoul 08826, Korea 7 Department of Industrial Machinery Engineering, College of Life Science and Natural Resources, Sunchon National University, Sunchon 57922, Korea 8 Interdisciplinary Program in IT-Bio Convergence System, Sunchon National University, Suncheon 57922, Korea * Correspondence: [email protected] (H.S.); [email protected] (P.G.); [email protected] (J.H.C.) † These authors contributed equally to the work. Abstract: Cisplatin, a representative anticancer drug used to treat cancer, has many adverse effects. In particular, it causes significant damage to the kidneys. -
Insights on the Interaction of Calcein with Calcium Carbonate and Its Implications in Biomineralization Studies
Insights on the interaction of calcein with calcium carbonate and its implications in biomineralization studies Giulia Magnabosco a,*, Iryna Polishchuk b, Jonathan Erez c, Simona Fermani a, Boaz Pokroy b, Giuseppe Falini a,* The effects of calcein, a marker commonly used to assess entrapment of calcein within the crystal structure of calcium mineral growth in calcifying organism, on calcite and aragonite carbonates is known to occur due its chemical structure.21,22 structure have been investigated. Calcein is entrapped within In this work, we examined in vitro the effect of calcein on the calcite and aragonite and modifies the shape and morphology growth of calcite and aragonite crystals, the two main 2+ of both polymorphs. Moreover, in the presence of Mg , it polymorphs of CaCO3 found in living organisms, using the inhibits aragonite formation in favor of magnesium calcite. calcein concentration in the range usually adopted for in vivo labelling. Studying and understanding of the biomineralization processes For this goal, we used the vapor diffusion method, consisting employ various methods to mark the growth of the inorganic in the diffusion of NH3(g) and CO2(g) obtained from the 2+ components of the organisms, allowing researchers to decomposition of (NH4)2CO3(s), into a Ca solution containing correlate a particular region of the skeleton to the instant of its the dye. This method is relevant for biomineralization process 2- deposition. A common method is to use fluorescent molecules due to the slow increase of the concentration of CO3 ions in to label a particular stage of the deposition process.1 Calcein, a the crystallization solution. -
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. -
Product Information Sheet
AAT Bioquest®, Inc. Product Technical Information Sheet Last Updated July 2012 Classic Calcium Detection Reagents Calcium acts as a universal second messenger in a variety of cells. Numerous functions of all types of cells are regulated by Ca2+ to a greater or lesser degree, thus calcium measurement is critical for numerous biological investigations. Since the 1920s, scientists have attempted to measure Ca2+, but few were successful due to limited availability of Ca2+ probes. The first reliable measurement of Ca2+ was performed by Ridgway and Ashley by injecting the photoprotein aequorin into the giant muscle fiber of the barnacle. Subsequently, in the 1980s, Tsien and colleagues produced a variety of fluorescent indicators. Among them Indo-1, Fura-2, Fluo-3 and Rhod-2 have been the most valuable dyes for measuring Ca2+ with a fluorescence instrument. Fluorescent probes that show spectral responses upon binding to Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Most of these fluorescent indicators are derivatives of BAPTA chelators that incorporate a PET system responsive to calcium. FLIPR® and FlexStation™ instruments of Molecular Devices Corp., FDSS of Hamamatsu Corp. and NOVOstar™ of BMG Technologies have enabled high throughput measurement of calcium for GPCR and ion channel research. There are quite a few factors that need to be considered when selecting a fluorescent Ca2+ indicator. Spectral Properties: For UV excitation, Indo-1 and Fura-2 are widely used. Fluo-3 is preferred for 488 nm excitation while Rhod-2 and X-rhod are used for red emissions. -
Instructions Calcein AM Cell Viability
IFU0211 Rev 1 Status: RELEASED printed 12/9/2016 11:41:26 AM by Trevigen Document Control Instructions For Research Use Only. Not For Use In Diagnostic Procedures Calcein AM Cell Viability Kit Catalog# 4892-010-K 1000 Tests* *Calculated based on using 1 μM final concentration of Calcein AM; Total number of tests varies with the concentration of Calcein AM required for particular cells. IFU0211 Rev 1 Status: RELEASED printed 12/9/2016 11:41:26 AM by Trevigen Document Control Calcein AM Cell Viability Kit Cat# 4892-010-K Table of Contents Page I. Introduction 1 II. Precautions and Limitations 2 III. Materials Supplied 2 IV. Materials/Equipment Required but not Supplied 2 V. Reagent Preparation 2 VI. Assay Protocol 3 VII. Standardization 4 VIII. Troubleshooting 5 IX. References 5 X. Related Products Available from Trevigen 5 © 2011 Trevigen, Inc. All rights reserved. Trevigen and TACS are registered trademarks and, FlowTACS, TiterTACS, MitoShift and DePsipher are trademarks of Trevigen, Inc. TACS: Trevigen Apoptotic Cell System i E2/25/11v1 IFU0211 Rev 1 Status: RELEASED printed 12/9/2016 11:41:26 AM by Trevigen Document Control I. Introduction Trevigen’s Calcein AM Cell Viability Kit provides a simple, rapid and accurate method to measure cell viability and/or cytotoxicity. Calcein AM (structure A) is a non-fluorescent, hydrophilic compound that easily permeates intact, live cells. The hydrolysis of Calcein AM by intracellular esterases produces calcein (structure B), a hydrophilic, strongly fluorescent compound that is well-retained in the cell cytoplasm. Cells grown in black-walled plates can be stained and quantified in less than two hours.