Analysis of Nucleic Acids Using Flow Cytometry
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Supplementary Data for Publication
Electronic Supplementary Material (ESI) for Physical Chemistry Chemical Physics. This journal is © the Owner Societies 2016 Supplementary Data for Publication Synthesis of Eucalyptus/Tea Tree Oil Absorbed Biphasic Calcium phosphate-PVDF Polymer Nanocomposite Films: A Surface Active Antimicrobial System for Biomedical Application Biswajoy Bagchi1,δ, Somtirtha Banerjee1, Arpan Kool1, Pradip Thakur1,2, Suman Bhandary3, Nur Amin Hoque1 , Sukhen Das1+* 1Physics Department, Jadavpur University, Kolkata-700032, India. 2Department of Physics, Netaji Nagar College for Women, Kolkata-700092, India. 3Division of Molecular Medicine, Bose Institute, Kolkata-700054, India. +Present Address: Department of Physics, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, West Bengal-711103, India. §Present Address: Fuel Cell and Battery Division, Central Glass and Ceramic Research Institute, Kolkata-700032, India. *Corresponding author’s email id: [email protected] Contact: +919433091337 Antimicrobial activity of EU and TTO treated films on E .coli and S. aureus by acridine orange/ethidium bromide (AO/EB) dual staining Live/dead cell characterization of EU/TTO film treated bacterial cultures was also done to visualize the viability under fluorescence microscope (). The treated culture suspensions after 12 and 24 hours of incubation were collected by centrifugation (5000 rpm, 20 mins). The cell pellets were resuspended in PBS. The staining solution was prepared by mixing equal parts of acridine orange (5mg/mL) and ethidium bromide (3mg/mL) in ethanol. 20μL of the staining solution is then mixed with 10μL of the resuspended solution and incubated for 15 minutes at 37°C. 10μL of this solution was then placed on a glass slide and covered with cover slip to observe under fluorescence microscope. -
Introduction to Flow Cytometry Principles Data Analysis Protocols Troubleshooting
Flow Cytometry ipl.qxd 11/12/06 11:14 Page i Introduction to Flow Cytometry Principles Data analysis Protocols Troubleshooting By Misha Rahman, Ph.D. Technical advisors Andy Lane, Ph.D. Angie Swindell, M.Sc. Sarah Bartram, B.Sc. Your first choice for antibodies! Flow Cytometry ipl.qxd 11/12/06 11:14 Page ii Flow Cytometry ipl.qxd 11/12/06 11:14 Page iii Introduction to Flow Cytometry Principles Data analysis Protocols Troubleshooting By Misha Rahman, Ph.D. Technical advisors Andy Lane, Ph.D. Angie Swindell, M.Sc. Sarah Bartram, B.Sc. Flow Cytometry ipl.qxd 11/12/06 11:14 Page 2 Preface How can I explain what flow cytometry is to someone that knows nothing about it? Well, imagine it to be a lot like visiting a supermarket. You choose the goods you want and take them to the cashier. Usually you have to pile them onto a conveyor. The clerk picks up one item at a time and interrogates it with a laser to read the barcode. Once identified and if sense prevails, similar goods are collected together e.g. fruit and vegetables go into one shopping bag and household goods into another. Now picture in your mind the whole process automated; replace shopping with biological cells; and substitute the barcode with cellular markers – welcome to the world of flow cytometry and cell sorting! We aim to give you a basic overview of all the important facets of flow cytometry without delving too deeply into the complex mathematics and physics behind it all. For that there are other books (some recommended at the back). -
Comparison of Count Reproducibility, Accuracy, and Time to Results
Comparison of Count Reproducibility, Accuracy, and Time to Results between a Hemocytometer and the TC20™ Automated Cell Counter Tech Frank Hsiung, Tom McCollum, Eli Hefner, and Teresa Rubio Note Bio-Rad Laboratories, Inc., Hercules, CA 94547 USA Cell Counting Bulletin 6003 Introduction Bead counts were performed by sequentially loading For over 100 years the hemocytometer has been used by and counting the same chamber of a Bright-Line glass cell biologists to quantitate cells. It was first developed for the hemocytometer (Hausser Scientific). This was repeated ten quantitation of blood cells but became a popular and effective times. The number of beads was recorded for all nine tool for counting a variety of cell types, particles, and even 1 x 1 mm grids. small organisms. Currently, hemocytometers, armed with Flow Cytometry improved Neubauer grids, are a mainstay of cell biology labs. Flow cytometry was performed using a BD FACSCalibur flow Despite its longevity and versatility, hemocytometer counting cytometer (BD Biosciences) and CountBright counting beads suffers from a variety of shortcomings. These shortcomings (Life Technologies Corporation). Medium containing 50,000 include, but are not limited to, a lack of statistical robustness at CountBright beads was combined one-to-one with 250 µl low sample concentration, poor counts due to device misuse, of cells in suspension, yielding a final solution containing and subjectivity of counts among users, in addition to a time- 100 beads/µl. This solution was run through the flow consuming and tedious operation. In recent years automated cytometer until 10,000 events were collected in the gate cell counting has become an attractive alternative to manual previously defined as appropriate for non-doublet beads in hemocytometer–based cell counting, offering more reliable the FSC x SSC channel. -
The Past, Present and Future of Flow Cytometry in Central Nervous System Malignancies
Review The Past, Present and Future of Flow Cytometry in Central Nervous System Malignancies Evrysthenis Vartholomatos 1, George Vartholomatos 2, George A. Alexiou 1,3 and Georgios S. Markopoulos 1,2,* 1 Faculty of Medicine, Neurosurgical Institute, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece; [email protected] (E.V.); [email protected] (G.A.A.) 2 Haematology Laboratory-Unit of Molecular Biology, University Hospital of Ioannina, 45110 Ioannina, Greece; [email protected] 3 Department of Neurosurgery, University of Ioannina, 45110 Ioannina, Greece * Correspondence: [email protected] Abstract: Central nervous system malignancies (CNSMs) are categorized among the most aggressive and deadly types of cancer. The low median survival in patients with CNSMs is partly explained by the objective difficulties of brain surgeries as well as by the acquired chemoresistance of CNSM cells. Flow Cytometry is an analytical technique with the ability to quantify cell phenotype and to categorize cell populations on the basis of their characteristics. In the current review, we summarize the Flow Cytometry methodologies that have been used to study different phenotypic aspects of CNSMs. These include DNA content analysis for the determination of malignancy status and phenotypic characterization, as well as the methodologies used during the development of novel therapeutic agents. We conclude with the historical and current utility of Flow Cytometry in the field, and we propose how we can exploit current and possible future methodologies in the battle against this dreadful type of malignancy. Citation: Vartholomatos, E.; Keywords: central nervous system malignancies; flow cytometry; glioblastoma; intraoperative flow Vartholomatos, G.; Alexiou, G.A.; cytometry; phenotypic analysis; DNA content analysis Markopoulos, G.S. -
Preliminary Experiment for Cell Count Using Flow Cytometry1 Yuki Morono,2 Jens Kallmeyer,2 Fumio Inagaki,2 and the Expedition 329 Scientists2
D’Hondt, S., Inagaki, F., Alvarez Zarikian, C.A., and the Expedition 329 Scientists Proceedings of the Integrated Ocean Drilling Program, Volume 329 Preliminary experiment for cell count using flow cytometry1 Yuki Morono,2 Jens Kallmeyer,2 Fumio Inagaki,2 and the Expedition 329 Scientists2 Chapter contents Abstract Abstract . 1 One of the major challenges in microbial ecology is to evaluate the accurate number of living cells in a natural environment. Cell Introduction . 1 count using flow cytometry is a powerful, high-throughput tech- Principles of fluorescent wavelength–based cell discrimination. 2 nique that has widely been used for aquatic habitats but not for sedimentary environments because mineral grains interfere with Standardization of flow cytometry cell count protocol for South Pacific Gyre sediment . 2 cell detection. During Integrated Ocean Drilling Program Expedi- Results and discussion. 3 tion 329, we tested several sample preparation methods for on- board cell counting using flow cytometry with various pelagic Conclusion and future prospects . 6 sediments from the South Pacific Gyre. The cell numbers acquired Acknowledgments. 6 from shallow sediments are almost consistent with microscopic References . 6 direct counts (i.e., ~105 cells/cm3). Yet, the method needs im- Figures . 7 provement and standardization to reduce the background signal and to lower the minimum detection limit for deep sedimentary habitats with very low cell densities (i.e., <1000 cells/cm3). Introduction Detection and enumeration of microbial life in marine subsurface environments provides primary information for deciphering the extent and habitability of Earth’s biosphere. Hence, accurate and precise knowledge of microbial cell abundance at high spatial res- olution is a major challenge for the exploration of subsurface life. -
Novel Acridine Orange Staining Protocol and Microscopy with UV Surface Excitation Allows for Rapid Histological Assessment of Canine Cutaneous Mast Cell Tumors
Novel Acridine Orange Staining Protocol and Microscopy with UV Surface Excitation Allows for Rapid Histological Assessment of Canine Cutaneous Mast Cell Tumors Croix Griffin1, Richard Levenson2, Farzad Feredouni2, Austin Todd2 1. School of Veterinary Medicine, University of California, Davis 2. Department of Pathology and Laboratory Medicine, University of California, Davis Compression Sponge Tissue Introduction ❖ XYZ Stage Stained for 1 minute UV window ❖ Mast cell tumors (MCTs) represent up to 27% of all malignant cutaneous tumors in with 0.03% Acridine dogs. Diagnosis is easily made with cytology, however the grade of the tumor LED: 1 Orange titrated to determines prognosis and required margins. UV excitation pH 0.53 with HCl ~280 nm ❖ Grade is not typically known until at least 48 hours after the surgery, as pre-surgical incisional biopsies are impractical due to additional cost and risk to the ❖ Rinsed in phosphate Objective patient. 5, 10, or 20X buffered saline for 1 ❖ Up to 30% of all mid to high grade mast cell tumors recur when incompletely minute at 50°C Tube Lens excised. Recurrence is correlated with progression to malignancy in 59% of patients.2 Figure 1. Example of MCT removal surgery. Figure 2. Blue arrow route does Figure 3. A streamlined, robust, Figure 4. MUSE Microscope. Please ❖ Low grade tumors are unlikely to recur regardless of margin cleanliness.3 When Single or multiple biopsies can be cut by hand not require formalin fixation or yet relatively gentle MC staining visit www.musepathology.com for removed with wide margins, the patient endures unnecessary short term morbidity from the tumor bed or tumor itself. -
ACRIDINE ORANGE STAIN - for in Vitro Use Only - Catalogue No
ACRIDINE ORANGE STAIN - For in vitro use only - Catalogue No. SA16 Our Acridine Orange Stain is used as a Quality Control fluorescent staining agent to detect the presence of bacteria in blood cultures and other bodily fluids. After checking for correct pH, colour, depth, Acridine orange is a fluorochrome dye that and sterility, the following organisms are used to can interchalate into nucleic acid. At a low pH determine the growth performance of the under UV light, bacterial and fungal nucleic acid completed medium. fluoresces orange whereas background mammalian nucleic acid fluoresces green. This Organism Expected Results rapid fluorescent staining procedure has been reported to be more sensitive than the Gram Escherichia coli Orange fluorescence staining procedure in the detection of ATCC 25922 microorganisms in blood cultures, cerebral spinal fluid and buffy coat preparations. Acridine orange stain can also aid in the detection of Storage and Shelf Life Acanthamoeba infections, infectious keratitis, Helicobacter pylori gastritis, and cell wall Our Acridine Orange Stain should be stored in deficient-bacteria such as Mycoplasma . the upright position at room temperature. Under these conditions this medium has a shelf life of 52 Formulation per Litre weeks from the date of manufacture. Acridine Orange ......................................... 100 mg Acetate Buffer .......................................... 1000 mL Ordering Information pH 4.0 ± 0.2 Cat# Description Format SA16-250 Acridine Orange Stain 250-mL Each Recommended Procedure 1. The prepared slide is fixed in methanol and air-dried. References 2. Flood the slide with Acridine Orange Stain. Allow the stain to sit on the slide for 2 1. Lauer BA, Reller LB, Mirrett S. -
Binding of Acridine Orange to DNA in Situ of Cells from Patients with Acute Leukemia1
(CANCER RESEARCH 49. .1692-3695. July 1. 1989] Binding of Acridine Orange to DNA in Situ of Cells from Patients with Acute Leukemia1 Alexander J. Walle2 and George Y. Wong Cornell Õ'nirersily Medical (allege /A. J. H'./and Memorial Sloan-Kettering Cancer Ccaler ¡G.Y. W.], New York, New York 10021 ABSTRACT cytes bound AO in a manner significantly different from that of human leukemic blood lymphoblasts [L3 variety of the Fluorescence flow cytometry was used to generate DNA histograms of French-American-British classification (6)]. We quantitatively at nilim- orange stained leukemic cell populations in G(I-G| phase of the described the interaction of AO with DNA of leukemic cell cell cycle. Complexes of the intercalating agent, acridine orange, with populations in G0-G| phase of the cell cycle by measuring the double-stranded DNA in situ, emit green fluorescence upon excitation with blue laser light. The histograms were evaluated by tirsi determining standard deviation of green fluorescence intensity pulses emit ted from AO-DNA complexes in single cells around the mean the standard deviation of the fluorescence intensity relative to the mean channel of fluorescence, i.e., the coefficient of variation, and then dividing channel of the fluorescence histograms. This represents the CV the coefficient of variation of a patient's sample by that of a control of the fluorescence intensity histograms (Fig. 1). To standardize sample (rCV). The mean rCV of cell populations of acute lymphoblastic and calculate the CVs of patient cell histograms, and subse leukemia (31 patients) differed significantly from that of nonlymphoblas- quently to analyze the quantitative differences between the tic leukemia (21 patients). -
A Deep Profiler's Guide to Cytometry
TREIMM-937; No. of Pages 10 Review A deep profiler’s guide to cytometry 1 1 2 2 Sean C. Bendall , Garry P. Nolan , Mario Roederer and Pratip K. Chattopadhyay 1 Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA 2 ImmunoTechnology Section, Vaccine Research Center, NIAID, NIH, Bethesda, MD, USA In recent years, advances in technology have provided us and data analysis, and have led to state-of-the-art 20-pa- with tools to quantify the expression of multiple genes in rameter flow cytometers. Concomitant with this develop- individual cells. The ability to measure simultaneously ment, our understanding of immunology and stem cell multiple genes in the same cell is necessary to resolve biology has matured tremendously with the discovery of the great diversity of cell subsets, as well as to define scores of functionally diverse cell populations. Here, we their function in the host. Fluorescence-based flow cyto- review the development and highlight applications of poly- metry is the benchmark for this; with it, we can quantify chromatic flow cytometry (PFC, 6+ colors). In addition, we 18 proteins per cell, at >10 000 cells/s. Mass cytometry is review recent advances in a next-generation, ‘post-fluores- a new technology that promises to extend these capa- cence’ single-cell technology termed mass cytometry, which bilities significantly. Immunophenotyping by mass spec- is theoretically capable of measuring 70–100 parameters. trometry provides the ability to measure >36 proteins at Both fluorescence and mass cytometry have unique and a rate of 1000 cells/s. -
AO/EB Staining Kit
AO/EB Staining Kit Cat.No.: E607308 Package: 100 Tests/200 Tests Description Acridine orange (AO) is a nucleic acid selective fluorescent cationic dye. It is cell-permeable, and will stain both live and dead cells. AO is commonly used for fluorescence microscopy and flow cytometry analysis of cellular physiology and cell cycle status. This cell-permeant cellular stain can be utilized in conjunction with a number of other staining solutions. Under the fluorescent microscope: Live cells will appear uniformly green; Apoptotic cells will stain green and contain bright green dots in the nuclei as a consequence of chromatin condensation and nuclear fragmentation; Necrotic cells will stain orange, but the fluorescent is weak or even disappear. EB can stain only cells that have lost membrane integrity. Combined with EB, necrotic cells stain orange, but have a nuclear morphology resembling that of viable cells, with no condensed chromatin. Then normal cells, apoptotic cells and necrotic cells can be distinguished by using this AO/EB staining kit. Kit Components Component 100 Tests 200 Tests Component A: AO Staining Solution 0.5 ml 1 ml Component B: EB Staining Solution 0.5 ml 1 ml Component C: 10X Buffer 10 ml 20 ml Protocol 1 1 Storage Store at 2~8°C and protect from light. Reagent is stable for at least one year. Procedure Note: The follow procedure is adapted for most cell types. 1. Dilute 10X Buffer (Component C) with distilled water to 1X Buffer. 2. Wash cells twice with phosphate buffer saline (PBS) and re-suspend cells in desired volume of 1X Buffer. -
PGMD/Curcumin Nanoparticles for the Treatment of Breast Cancer Mankamna Kumari1, Nikita Sharma1, Romila Manchanda2, Nidhi Gupta3, Asad Syed4, Ali H
www.nature.com/scientificreports OPEN PGMD/curcumin nanoparticles for the treatment of breast cancer Mankamna Kumari1, Nikita Sharma1, Romila Manchanda2, Nidhi Gupta3, Asad Syed4, Ali H. Bahkali4 & Surendra Nimesh1* The present study aims at developing PGMD (poly-glycerol-malic acid-dodecanedioic acid)/curcumin nanoparticles based formulation for anticancer activity against breast cancer cells. The nanoparticles were prepared using both the variants of PGMD polymer (PGMD 7:3 and PGMD 6:4) with curcumin (i.e. CUR NP 7:3 and CUR NP 6:4). The size of CUR NP 7:3 and CUR NP 6:4 were found to be ~ 110 and 218 nm with a polydispersity index of 0.174 and 0.36, respectively. Further, the zeta potential of the particles was − 18.9 and − 17.5 mV for CUR NP 7:3 and CUR NP 6:4, respectively. The entrapment efciency of both the nanoparticles was in the range of 75–81%. In vitro anticancer activity and the scratch assay were conducted on breast cancer cell lines, MCF-7 and MDA-MB-231. The IC50 of the nanoformulations was observed to be 40.2 and 33.6 μM at 48 h for CUR NP 7:3 and CUR NP 6:4, respectively, in MCF-7 cell line; for MDA-MB-231 it was 43.4 and 30.5 μM. Acridine orange/EtBr and DAPI staining assays showed apoptotic features and nuclear anomalies in the treated cells. This was further confrmed by western blot analysis that showed overexpression of caspase 9 indicating curcumin role in apoptosis. With the changing environmental factors and lifestyle such as pollution, tobacco smoking, diet patterns, there has been a tremendous increase in the incidence of cancer. -
Quantitative Phase Imaging in Biomedicine
REVIEW ARTICLE https://doi.org/10.1038/s41566-018-0253-x Quantitative phase imaging in biomedicine YongKeun Park1,2,3, Christian Depeursinge4,5 and Gabriel Popescu 6* Quantitative phase imaging (QPI) has emerged as a valuable method for investigating cells and tissues. QPI operates on unla- belled specimens and, as such, is complementary to established fluorescence microscopy, exhibiting lower phototoxicity and no photobleaching. As the images represent quantitative maps of optical path length delays introduced by the specimen, QPI provides an objective measure of morphology and dynamics, free of variability due to contrast agents. Owing to the tremen- dous progress witnessed especially in the past 10–15 years, a number of technologies have become sufficiently reliable and translated to biomedical laboratories. Commercialization efforts are under way and, as a result, the QPI field is now transi- tioning from a technology-development-driven to an application-focused field. In this Review, we aim to provide a critical and objective overview of this dynamic research field by presenting the scientific context, main principles of operation and current biomedical applications. maging biological cells and tissues is central to biological research will wash out the image altogether due to multiple scattering. Both and medical diagnosis. Following its four-century-old history, of these extremes result in poor image contrast. Single cells and thin microscopy has become the most commonly used tool in medi- tissue slices belong to the first category: the scattered light they gen- I 1 cine and biology . However, despite significant breakthroughs, opti- erate is orders of magnitude weaker than the incident light.