A2780/DDP-R. Carmen Raventos-Suarez Ph.D. ( ) Bristol-Myers Squibb
|
|
- Naomi Green
- 7 years ago
- Views:
Transcription
1 A2780/DDP-R Control nM Carmen Raventos-Suarez Ph.D. ( ) Bristol-Myers Squibb
2 When We Study Cells At the microscope we can visualize many structures inside the cells FACS Answers presented on this workshop Proliferation: What When and How Arrest: Why When is a key feature Apoptosis: Dealing with the kinetics of a disappearing population Photographs from Molecular Probes web site
3 A2780/DDP-R Proliferation What? A cycling phenomena regulated by check point controls DNA duplication followed by segregation into two new entities Control nM
4 Proliferation When do you measure it? Basic science research signal transduction evaluation Pharmaceutical industry biochemical to cellular assays Clinical follow up cancer therapeutic efficiencies inflammatory cascades identification immune activation responses
5 Cell Cycle Profile Components A picture of DNA Synthesis G0/G1 Cell Quest software S G2/M Sub-G1 Tetraploid DNA (area)
6 Cell Cycle Profiles: A Personal Signature of Cells DNA histograms gives a first hint about cells proliferation potential Slow growth Fast growth DNA-PI
7 Proliferation Features What does Cell Cycle Profiles really mean? A2780WT A2780DDP Confluent Confluent FL2-A: PI Sparse Logaritmic FL2-A: PI Sparse Logaritmic FlowJo Software
8 Cell Cycle Times and Numbers DNA content and population distributions 24h Number of cells =100 G1 50% S 30% G2/M 20% 24h Cell cycle 12hrs 7.2hrs 4.8hrs 24h Number of cells =125 G1 40% S 38% G2/M 22% new cycle 18h Cell cycle 7.2 hrs 6.8 hrs 3.9hrs 6h additional hours of growth
9 When A Cell Divides Addressing cells one at a time S G1 G2/M Modified from Cell Signaling Web site
10 Getting The Components Right The Question of Doublet Discrimination FlowJo Software Alternatively Area vs. Height is also a way to discriminate doublets and higher clumps
11 Cell Cycle Profile of Single Cells Gated vs. non gated populations Previous plots FlowJo Software # Cells single cells %G1 %S %G2 %<G1 %>G # Cells Single cells %G1 %S %G2 %<G1 %>G # Cells single cells %G1 %S %G2 %<G1 %>G FL2-A: DNA-PI 0 FL2-A: FL2-Area 0 FL3-A: DNA-7AAD # Cells Ungated %G1 %S %G2 %<G1 %>G # Cells Ungated %G1 %S %G2 %<G1 %>G # Cells Ungated %G1 %S %G2 %<G1 %>G FL2-A: DNA-PI 0 FL2-A: FL2-Area 0 FL3-A: DNA-7AAD
12 Nuclei vs. Whole Cells What is better? Nuclei Ploidy paraffin blocks Apoptosis problems Nuclear localization Whole cells Ploidy fresh samples Apoptosis efficiency Cytoplasmic markers.
13 Cell Cycle Software Packages Multicycle Modfit FlowJo Getting Numbers to Fit Your Data All are packages that contain algorithms with different restrictions bound to their calculations. Unfortunately, no algorithms are consistently reliable in fitting all the distributions you may encounter. You may have to experiment with different options and constraints in finding your best results. (copied FlowJo quote)
14 Getting To See The Numbers Watson Pragmatic vs. Dean Jett Fox in FlowJo software # Cells single %G1 %S %G # Cells single %G1 %S %G # Cells single %G1 %S %G FL2-A: DNA-PI 0 FL2-A: DNA-PI 0 FL2-A: DNA-PI 1000 single 400 single single # Cells %G1 %S %G # Cells %G1 %S %G # Cells %G1 %S %G FL2-A: DNA-PI 0 FL2-A: DNA-PI 0 FL2-A: DNA-PI Alice Givan previously showed the Mod Fit variations on their selective options
15 Precautions-Tissue Culture Monolayers Learn the metabonomics of your cells Doubling times, density optimization Cell Culture Stocks Maintenance Rigid protocol for split times/avoid overgrowth Protocol for Experimental Tests Standardized number of cells & scheduling Efficiently trypsinize to a unicellular suspension
16 Beyond DNA Quantitative Analysis How to add more significance to your data? Adding a second marker to your cells Cell identification markers (CD s or signal transduction probes) DNA doubling features, BrdU incorporation Adding a third or more markers to your cells When preset configuration is fixed in your instrument Search for probes that allow to you the best combinations on added color When you are able to set up your own configuration First pick up different lasers then change your dichroics and band filters
17 Proliferation and BrdU S phase and Doubling Times K min pulse 5hrs release 8hrs release FL1-H 100 FL1-H 100 FL1-H 100 FL1-H FL2-A A2780S FL2-A FL2-A FL2-A FlowJo software FL1-H 100 FL1-H 100 FL1-H 100 FL1-H FL2-A 1 FL2-A 1 FL2-A 1 FL2-A
18 Calculating the Numbers Getting the mathematics to work for you From: Cell Cycle Kinetics Estimated by Analysis of Bromodeoxyuridine Incorporation. Terry N.H.A., and White A. Methods in cell Biology 63: (1994)
19 Overall messages When to do DNA? Maybe every time you address cells Watch out! Single cells exclusively Cell preparation. Avoid clumping How to address specific questions? Cell culture conditions. Use tight protocols Most appropriate software. Up to you
20 References Solid Tumor dissociation and storage (nuclei) Cerra.R., Zarbo R.J., and Crissman. JD.,1990 Dissociation of cells from Solid Tumors. Methods in Cell biology 33: 1-12 Vindelov.L.L.,Cristensen.IJ. An integrated set of methods for routine flow cytometry DNA Analysis Methods in Cell biology 33: Cell Lines (whole cells) Pozarowwski.P., Darzynkiewiwicz.Z. Analysis of Cell Cycle by Flow Cytometry Methods Mol Biol 281: Traganos.F.,Juan.G.,Darzynkiewiwicz.Z. Cell Cycle Analysis of Drug treated Cells Methods Mol Biol 95: Overall Rabinovitch.P.S. Practical considerations for DNA Content and Cell Cycle Analysis in Clinical Flow Cytometry. Principles and applications. Williams and Wilkins-1993 Pham.NA., Jaccobberger.JW.,Schimmer.A.D.,Cao.P.,Gronda.M.,Hedley.D.W The dietary isothiocyanate sulforaphane targets pathways of apoptosis,cell cycle arrest, and oxidative stress in human pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficient mice. Mol.Cancer.Ther.3: Bagwell.B. et al Optimizing Flow Cytometric DNA ploidy and S phase Fraction as nnegative prosnostic Markers for Node-Negative Breast Cancer Specimens Cytometry 46:
21 A2780/DDP-R Proliferation Disturbances Arrest Control nM A Way to Cope with Stress
22 ARREST What is it? A slowdown in cell cycle progression? A reversible or irreversible block in one phase of the cycle? An induced effect of stress or toxic insult? A response of the cell genetic makeup? All of the above?
23 The Meaning of Cell Cycle Arrest How Do You Identify Specific Growth Arrest? Control Campthothecin Cisplatin Paclitaxel Cell growth arrest induced by stress or chemical compounds are efficiently identified when cells are growing at their optimal logarithmic rates and inducers are at an equilibrium concentration where cells are halted to activate repair mechanisms or apoptosis Cell Quest software
24 Identification of Arrest Taxol Paclitaxel a model drug for G2/M arrest DRUG-CELL CYCLE PROFILES AFTER 24HRS EXPOSURE A2780/DDP-S A2780/DDP-R Taxotere Titrations Control nM Control nM Laidlaw, J., Raventos-Suarez,C., Fairchild, C.R., Peterson, R.W., and Menendez, A.T. Taxol and taxotere have similar potency in cytotoxicity assays, cell selectivity, bcl-2 phosphorylation, G2/M arrest and induction of apoptosis.91 st Annual meeting of the AACR. San Francisco, April CellQuest software
25 Identification of Arrest Camptothecin a Model drug for S arrest? LnCaP Camptothecin A2780 Camptothecin 100nM 100nM 30nM 30nM FL2-A: DNA-PI 1nM Control none nM 10nM More titrations FL2-A: DNA-PI 1nM Control none nM 10nM FlowJo Software
26 How to Evaluate a Compound Preliminary Requirements Cytotoxic tests provide the best tool to identify dosage Cell Cycle Titration Curves Cell cycle profiles need to be address one cycle of growth at a time Preliminary titration curves done at a 24h time point provide good hints of activity
27 Arrest As An Assay The Story of BMS Mechanism of Action BMS is the first non camptothecin compound described to be specific for Topoisomerase I inhibition. Because of the side effects of camptothecin another efficient compound with this kind of activity have been actively pursued by the pharmaceutical industry Biochemical assays required confirmation at the cellular level to reinforce the value of this compound as a candidate for the clinic
28 Results on BMS BMS IC 50 = 3 nm Flow cytometry analysis of A2780 cells with wt p53 Camptothecin IC 50 = 10 nm Effects of BMS and camptothecin Control 0.1x 1x 5x 10x 20x IC50 CellQuest software -Raventos-Suarez,C., Class. K., Buczek,J,L.,Balasubramanian,N., Long, B., and Menendez, A,T Topoisomerase I is the Target Responsible for Cytotoxicity of BMS : Confirmation by Flow Cytometry 92th Annual Meeting, New Orleans March 24-28; Proc. AACR :103 Abs 562 -Raventos-Suarez,C., Class, K., Wild, R., Menendez, A., Long, B. 004 IN VITRO Specificity Profiling of Cellular Topoisomerase Activities Using FACS Analyses.2ISAC XXII International Congress on Analytical Cytology. May Montpellier, France. Cytometry 58A: p115 Abst# 96207
29 HCT116 HCT116(VM46) In Arrest Profiles HCT116 (VM46) DNA Cell Cycle Profiles 488nm laser Control cells A second parameter reinforced your observations 633nM laser Cyclin B1 Profiles DNA Cyclin B1 Cyclin E Cyclin A FL2-H: FL2-Height FL1-H: FL1-Height Camptothecin treated 0 0 FL4-A: FL4 A FL4-A: FL4 A FL2-H: FL2-Height FL1-H: FL1-Height FL4-A: FL4 A FL4-A: FL4 A Comparison between PI and ToPro3 Blue= 488nm Red=633nm
30 FlowJo Allows Line Graphs Over DNA Profiles A second parameter always reinforced your observations Cyclin E Cyclin A
31 Results on BMS We used a panel of 3 paired cell lines: sensitive and resistant to identify activity It takes several comparisons to efficiently confirm at the cellular level effects of known activities from a biochemical assay
32 A2780/DDP-R Arrest Leads into Apoptosis Control nM
33 Apoptosis An active metabolic process devised to dismiss stressed cells unable to cope with the insult An active act of disappearance
34 Morphology of Apoptosis Control Apoptotic
35 Apoptosis Pictures Apoptosis a disappearing population
36 Evaluation Methods Best When Linked to DNA profiles Annexin V TdT Tunel p85parp Caspase 3 Many other caspases Other approaches- The Sub G1 fraction
37 Tunel Assay Provides Specificity of Phase apoptosis single cells apoptosis single cells apoptosis single cells apoptosis single cells FL1-H: dutp-fitc 10 2 FL1-H: dutp-fitc 10 2 FL1-H: dutp-fitc 10 2 FL1-H: dutp-fitc FL2-A: DNA-PI FL2-A: DNA-PI FL2-A: DNA-PI FL2-A: DNA-PI Dot Plot Overlays Flow Jo software
38 p85parp on Drug Effects Localization of populations engaged in apoptosis Vinblastin 5nM Colchicine 10nM FL1-H: P85-FITC 100 FL1-H: P85-FITC FL2-A: PI-DNA 1 FL2-A: PI-DNA Flow Jo software
39 The Sub-G1 Fraction * * * 0 * Low dose 600 FL2-A: DNA-PI 800 Control 1000 Apoptosis by Sub-G1, an increasingly growing population High dose
40 Taxol A2780/DDP-R A2780/DDP-S Taxotere When Proliferation, Arrest and Apoptosis Meet Control nM Control nM
41 The Story of BMS BMS is a farnesyl transferase inhibitor with high apoptosis induction capabilities. Targets on the farnesyl transferase cascade are not expected to produce any apoptosis in short periods of time but this compound did it An assay needed to be devised to stain for proliferation and apoptosis simultaneously to directly answer this question -Raventos-Suarez,C., Class. K. and Lee F The pro-apoptotic FT-inhibitor BMS selectively targets nonproliferating tumor cells. Demonstration by a new flow cytometric method. ISAC XXI International Congress on Analytical Cytology.May 2002 San Diego, California. Cytometry supp11: p72
42 Building An Assay Proliferation by BrdU Uptake and evaluation by Dnase treatment: modify the BrdU Flow Kit from BD cat# Cell cycle profiles by DNA 7AAD stain Apoptosis by p85 PARP use the Anti-PARP p85 fragment Ab from Promega cat#g734a
43 Proliferation and Apoptosis A Mechanism of Action of BMS P+A A P+A A P+A P P P Control Cisplatin 500nM BMS uM Red = non proliferating Blue = proliferating Green = apoptotic non proliferating P+A = proliferating cells engaged in apoptosis
44 Take Home Messages Cell Cycle Profiles are the best handle in addressing cell behavior, capacity to respond to an stimulus and possible engagement in apoptosis Careful preparation of cells is essential Multiparameter analysis linked to cell cycle profiles provide a more complete picture of cellular activities
45 Back up data
46 Cell Cycle Profile of Cells Getting to see the numbers When algorithms don t seem to fit you can force constrains FlowJo Software
47 Cell Cycle Profile of Cells A cell specific signature Panel of cell lines growing at logarithmic growth rates FlowJo Software HCT116 HCT116(VP35) HCT116(VM46) FL2-A: PI 800 A2780/DDP-R A2780/Tax A2780/DDP-S 1000 Why are times at logarithmic rate of growth taken as optimal?
48 Results on BMS Six cell lines two time points plus two control drugs HCT116 (24h) HCT116 (48h) HCT116/TopoII (24h) HCT116/TopoII (48h) P388 (24h) P388 (48h) P388/Topo I (24h) P388/Topo I (48h) A2780mutp53 (24h) A2780mutp53 (48h) A2780mutp53 (24h) A2780mutp53 (48h) Camptothecin IC50= 10nM Camptothecin IC50= 10nM Camptothecin IC50= 10nM Camptothecin IC50= 10nM Camptothecin IC50= 10nM Camptothecin IC50= 10nM Etoposide IC50= 1.2uM Etoposide IC50= 1.2uM Etoposide IC50= 1.2uM Etoposide IC50= 1.2uM Etoposide IC50= 1.2uM Etoposide IC50= 1.2uM BMS IC50=3nM BMS IC50=3nM BMS IC50=3nM BMS IC50=3nM BMS IC50=3nM BMS IC50=3nM Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 It takes a panel of comparisons to address population distribution effects Shown are Cells in 3 pairs:sensitive/resistant (TopoII, TopoI and p53)
49 Proliferation What and When Beyond DNA quantitative analysis Proliferation activities require more than DNA measurements to account for disturbances BrdU assay will identify the S phase and determine doubling times. Mitotic markers: will allow discrimination of G2 and S phases Cyclins: will determine how far into a cell cycle phase cells have been able to go before stop growing Specific altered functions by induced by resistance or mutation mechanisms
50 More on Mechanism of Action Specificity on Topo II A Camptothecin Resistant Cell line Control Camptothecin Etoposide TAS-103 A Camptothecin Sensitive Cell line Control Camptothecin Etoposide TAS-103 Long, B.H., Fairchild, C.R., Raventos-Suarez,C., Cornell, L., and Menendez, A.T. Cytotoxic mechanism of TAS-103 is related to topoisomerase II mediated DNA cleavage. 91 st Annual meeting of the AACR. San Francisco, April 1-5, Cell Quest software
Cell Cycle Phase Determination Kit
Cell Cycle Phase Determination Kit Item No. 10009349 Customer Service 800.364.9897 * Technical Support 888.526.5351 www.caymanchem.com TABLE OF CONTENTS GENERAL INFORMATION 3 Materials Supplied 3 Safety
More informationCELL CYCLE BASICS. G0/1 = 1X S Phase G2/M = 2X DYE FLUORESCENCE
CELL CYCLE BASICS Analysis of a population of cells replication state can be achieved by fluorescence labeling of the nuclei of cells in suspension and then analyzing the fluorescence properties of each
More informationApplication Note No. 2 / July 2012. Quantitative Assessment of Cell Quality, Viability and Proliferation. System
Application Note No. 2 / July 2012 Quantitative Assessment of Cell Quality, Viability and Proliferation System Quantitative Assessment of Cell Quality, Viability and Proliferation Introduction In vitro
More informationUses of Flow Cytometry
Uses of Flow Cytometry 1. Multicolour analysis... 2 2. Cell Cycle and Proliferation... 3 a. Analysis of Cellular DNA Content... 4 b. Cell Proliferation Assays... 5 3. Immunology... 6 4. Apoptosis... 7
More informationCell Cycle Tutorial. Contents
Cell Cycle Tutorial Contents Experimental Requirements...2 DNA Dyes...2 Protocols...3 PI Parameter & Analysis Setup...4 PI Voltage Adjustments...6 7-AAD Parameter Setup...6 To-Pro3 Parameter Setup...6
More informationCELL CYCLE BASICS. G0/1 = 1X S Phase G2/M = 2X DYE FLUORESCENCE
CELL CYCLE BASICS Analysis of a population of cells replication state can be achieved by fluorescence labeling of the nuclei of cells in suspension and then analyzing the fluorescence properties of each
More informationIntroduction to Flow Cytometry
Outline Introduction to Flow Cytometry Basic Concept of Flow Cytometry Introduction to Instrument Subsystems Daisy Kuo Assistant Product Manager E-mail: daisy_kuo@bd.com BDBiosciences Application Examples
More informationInternational Beryllium Conference, Montreal, Canada March 10, 2005
Alternative Lymphocyte Proliferation Tests: BrdU and Flow Cytometry Based Tests International Beryllium Conference, Montreal, Canada March 10, 2005 Tim K. Takaro Department of Environmental and Occupational
More informationHP8 herbal formula selectively inhibits prostate cancer cell line proliferation by inducing G2/M cell cycle arrest.
HP8 herbal formula selectively inhibits prostate cancer cell line proliferation by inducing G2/M cell cycle arrest. Flowers A, Banbury L, Leach D and Waterman P. Centre for Phytochemistry and Pharmacology,
More informationuse. 3,5 Apoptosis Viability Titration Assays
Introduction Monitoring cell viability can provide critical insights into the effectiveness of biological protocols and the stability of biological systems including: the efficacy of cell harvesting procedures,
More informationFlow Cytometry. What is Flow Cytometry? What Can a Flow Cytometer Tell Us About a Cell? Particle Size. Flow = Fluid Cyto = Cell Metry = Measurement
What is Flow Cytometry? Flow Cytometry Basic Principle and Applications BD Biosciences Daisy Kuo (daisy_kuo@bd.com) Flow = Fluid Cyto = Cell Metry = Measurement A variety of measurements are made on cells,
More informationAnnexin V-EGFP Apoptosis Detection Kit
ab14153 Annexin V-EGFP Apoptosis Detection Kit Instructions for Use For the rapid, sensitive and accurate measurement of apoptosis in various samples This product is for research use only and is not intended
More informationFighting the Battles: Conducting a Clinical Assay
Fighting the Battles: Conducting a Clinical Assay 6 Vocabulary: In Vitro: studies in biology that are conducted using components of an organism that have been isolated from their usual biological surroundings
More informationa Beckman Coulter Life Sciences: White Paper
a Beckman Coulter Life Sciences: White Paper Violet-Excited nim-da Allows Efficient and Reproducible Cell Cycle Analysis on the Gallios Flow Cytometer Authors: Valdez, Ben 1. Carr, Karen 2. Norman, John
More informationNotch 1 -dependent regulation of cell fate in colorectal cancer
Notch 1 -dependent regulation of cell fate in colorectal cancer Referees: PD Dr. Tobias Dick Prof. Dr. Wilfried Roth http://d-nb.info/1057851272 CONTENTS Summary 1 Zusammenfassung 2 1 INTRODUCTION 3 1.1
More informationab139418 Propidium Iodide Flow Cytometry Kit for Cell Cycle Analysis
ab139418 Propidium Iodide Flow Cytometry Kit for Cell Cycle Analysis Instructions for Use To determine cell cycle status in tissue culture cell lines by measuring DNA content using a flow cytometer. This
More informationAnnexin V-FITC Apoptosis Detection Kit
ab14085 Annexin V-FITC Apoptosis Detection Kit Instructions for Use For the rapid, sensitive and accurate measurement of Apoptosis in living cells (adherent and suspension). This product is for research
More informationCell Viability Assays: Microtitration (MTT) Viability Test Live/Dead Fluorescence Assay. Proliferation Assay: Anti-PCNA Staining
Cell Viability Assays: Microtitration (MTT) Viability Test Live/Dead Fluorescence Assay Proliferation Assay: Anti-PCNA Staining Spring 2008 1 Objectives To determine the viability of cells under different
More informationAnnexin V-FITC Apoptosis Detection Kit
ab14085 Annexin V-FITC Apoptosis Detection Kit Instructions for Use For the rapid, sensitive and accurate measurement of Apoptosis in living cells (adherent and suspension). This product is for research
More informationIslet Viability Assessment by Single Cell Flow Cytometry
Islet Viability Assessment by Single Cell Flow Cytometry Page 1 of 8 Purpose: To comprehensively assess the viability of the islet cell preparation prior to transplantation. Tissue Samples: A sample containing
More informationA. FSC and SSC gating of total BM cells. B. Gating strategy used to identify the Lin -
Supplementary Figure legends Figure S1. Multiparametric analysis of HSC Populations. A. FSC and SSC gating of total BM cells. B. Gating strategy used to identify the Lin - cell population. BM cells were
More informationFLOW CYTOMETRY: PRINCIPLES AND APPLICATIONS. By: Douaa Moh. Sayed
FLOW CYTOMETRY: PRINCIPLES AND APPLICATIONS By: Douaa Moh. Sayed Definition Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows
More informationFITC Annexin V/Dead Cell Apoptosis Kit with FITC annexin V and PI, for Flow Cytometry
FITC Annexin V/Dead Cell Apoptosis Kit with FITC annexin V and PI, for Flow Cytometry Catalog no. V13242 Table 1. Contents and storage information. Material Amount Composition Storage* Stability FITC annexin
More informationMarch 19, 2014. Dear Dr. Duvall, Dr. Hambrick, and Ms. Smith,
Dr. Daniel Duvall, Medical Officer Center for Medicare, Hospital and Ambulatory Policy Group Centers for Medicare and Medicaid Services 7500 Security Boulevard Baltimore, Maryland 21244 Dr. Edith Hambrick,
More informationPROTOCOL. Immunostaining for Flow Cytometry. Background. Materials and equipment required.
PROTOCOL Immunostaining for Flow Cytometry 1850 Millrace Drive, Suite 3A Eugene, Oregon 97403 Rev.0 Background The combination of single cell analysis using flow cytometry and the specificity of antibody-based
More informationFlow Cytometry Workshop:
Flow Cytometry Workshop: Section III: DNA Content Analysis Beckman Coulter Taiwan Branch 技 術 支 援 經 理 楊 人 泰 DNA Content Analysis-common sense DNA content analysis is one of the oldest Flow applications.
More informationBottlenecks in Clinical Source Material Acquisition. Aby J. Mathew, PhD May 5, 2009 ISCT Annual Meeting San Diego, CA amathew@biolifesolutions.
Bottlenecks in Clinical Source Material Acquisition Aby J. Mathew, PhD May 5, 2009 ISCT Annual Meeting San Diego, CA amathew@biolifesolutions.com Biopreservation What s the issue? Biopreservation considerations
More informationapoptosis detection Apoptosis assays for the Attune Acoustic Focusing Cytometer Apoptosis detection Attune Acoustic Focusing Cytometer
apoptosis detection ttune coustic Focusing Cytometer poptosis detection poptosis assays for the ttune coustic Focusing Cytometer poptosis is a carefully regulated process of cell death that occurs as a
More informationIntroduction to flow cytometry
Introduction to flow cytometry Flow cytometry is a popular laser-based technology. Discover more with our introduction to flow cytometry. Flow cytometry is now a widely used method for analyzing the expression
More informationApplication Note. Cell Cycle and DNA Content Analysis Using the BD Cycletest Assay on the BD FACSVerse System. Summary
Cell Cycle and DNA Content Analysis Using the BD Cycletest Assay on the BD FACSVerse System Yibing Wang, Catherine McIntyre, and Dev Mittar BD Biosciences, San Jose Contents 1 Summary 2 Introduction 3
More informationThe Need for a PARP in vivo Pharmacodynamic Assay
The Need for a PARP in vivo Pharmacodynamic Assay Jay George, Ph.D., Chief Scientific Officer, Trevigen, Inc., Gaithersburg, MD For further infomation, please contact: William Booth, Ph.D. Tel: +44 (0)1235
More informationContact Information Korea Health Industry Development Institute
AGM-130, a novel CDK inhibitor with a marked anti-tumor activity and reduced toxicity, targeting the triple negative breast cancer(tnbc) and tamoxifen-resistant estrogen receptor (ER) positive breast cancer
More informationKatharina Lückerath (AG Dr. Martin Zörnig) adapted from Dr. Jörg Hildmann BD Biosciences,Customer Service
Introduction into Flow Cytometry Katharina Lückerath (AG Dr. Martin Zörnig) adapted from Dr. Jörg Hildmann BD Biosciences,Customer Service How does a FACS look like? FACSCalibur FACScan What is Flow Cytometry?
More informationMOLECULAR PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
MOLECULAR PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS R. M. Weinshilboum, M.D., Program Director L. Wang, M.D., Ph.D., Program Co-Director D. C. Mays, Ph.D., Associate Program Director Ph.D. Degree Course
More information標 準 納 期 2 週 間 を 誇 るKINOMEscan 受 託 試 験 : 報 告 書 から 抽 出 できる 更 なるデータ? 日 本 事 業 担 当 大 嶺 青 河 DiscoveRx Corporation
標 準 納 期 2 週 間 を 誇 るKINOMEscan 受 託 試 験 : 報 告 書 から 抽 出 できる 更 なるデータ? 日 本 事 業 担 当 大 嶺 青 河 DiscoveRx Corporation DiscoveRx Corporation Fremont, CA San Francisco, CA San Diego, CA DiscoveRx Corporation 3 KINOMEscan
More informationBasic DNA Measurement by Flow Cytometry
Basic DNA Measurement by Flow Cytometry Ian Brotherick, PhD DNA, or deoxyribonucleic acid, is the molecule that carries the genetic information for all living organisms and is the main constituent of chromosomes.
More informationSteatosis Colorimetric Assay Kit
Steatosis Colorimetric Assay Kit Item No. 10012643 www.caymanchem.com Customer Service 800.364.9897 Technical Support 888.526.5351 1180 E. Ellsworth Rd Ann Arbor, MI USA TABLE OF CONTENTS GENERAL INFORMATION
More informationSafe Nano Design Molecule Manufacturing Market
August 14 16, 212 College of Nanoscale Science & Engineering (CNSE) of the University at Albany National Institute for Occupational Safety and Health (NIOSH) Prevention through Design Program Safe Nano
More informationKnipholone anthrone from Kniphofia foliosa induces a rapid onset of necrotic cell death in cancer cells
Knipholone anthrone from Kniphofia foliosa induces a rapid onset of necrotic cell death in cancer cells Fitoterapia, 11/18/2010 Habtemariam S The present study examines the comparative cytotoxicity of
More informationClassical drug resistance in the molecular age
Classical drug resistance in the molecular age Robert O Connor, Ph.D. Senior Programme Leader, Translational Cancer Pharmacology Lecturer in Biological Sciences, School of Nursing Chair ICORG Translational
More informationIntroduction to Flow Cytometry
Introduction to Flow Cytometry presented by: Flow Cytometry y Core Facility Biomedical Instrumentation Center Uniformed Services University Topics Covered in this Lecture What is flow cytometry? Flow cytometer
More informationMEASURABLE PARAMETERS: Flow cytometers are capable of measuring a variety of cellular characteristics such as:
INTRODUCTION Flow Cytometry involves the use of a beam of laser light projected through a liquid stream that contains cells, or other particles, which when struck by the focused light give out signals
More informationHow To Use Berberine
Programa Cooperación Farma-Biotech Jornada II: Oncología Novel Berberine derivatives as antitumor agents for cancer Barcelona, 13 de abril de 2011 Programa Cooperación Farma-Biotech Jornada II: Oncología
More informationAnimal Cell Culture. Third Edition. A Practical Approach OXJORD VNIVVRSITY 1'RVSS
Animal Cell Culture Third Edition A Practical Approach Edited by John R. W. Masters 3rd Floor Research Laboratories, University College London OXJORD VNIVVRSITY 1'RVSS Contents List of protocols page xiii
More informationCell, Tissue and Tumor Kinetics
Cell, issue and umor Kinetics Proliferation Kinetics: rate of growth of a population, change in total cell number. Adult tissues are in homeostasis. Children (and tumors) grow. I. Quantitative Assessment
More informationTechnical Bulletin. Threshold and Analysis of Small Particles on the BD Accuri C6 Flow Cytometer
Threshold and Analysis of Small Particles on the BD Accuri C6 Flow Cytometer Contents 2 Thresholds 2 Setting the Threshold When analyzing small particles, defined as particles smaller than 3.0 µm, on the
More informationCFSE Cell Division Assay Kit
CFSE Cell Division Assay Kit Item No. 10009853 Customer Service 800.364.9897 * Technical Support 888.526.5351 www.caymanchem.com TABLE OF CONTENTS GENERAL INFORMATION 3 Materials Supplied 4 Precautions
More informationRole Of Mitochondria In Mesenchymal Stem Cells
Role Of Mitochondria In Mesenchymal Stem Cells Roman Eliseev, MD, PhD, Tamara Raymond, Regis O'Keefe, MD, PhD. University of Rochester, Rochester, NY, USA. Disclosures: R. Eliseev: None. T. Raymond: None.
More informationExelixis Showcases R&D Pipeline at JPMorgan Healthcare Conference
Exelixis Showcases R&D Pipeline at JPMorgan Healthcare Conference Two New Clinical Programs and Significant Expansion of Cancer Pipeline Planned for 2004 SOUTH SAN FRANCISCO, Calif., Jan. 13 /PRNewswire-FirstCall/
More informationINSIDE THE BLACK BOX
FLOW CYTOMETRY ESSENTIALS INSIDE THE BLACK BOX Alice L. Givan Englert Cell Analysis Laboratory of the Norris Cotton Cancer Center Dartmouth Medical School HOW NOT TO BE A FLOW CYTOMETRIST Drawing by Ben
More informationCyTOF2. Mass cytometry system. Unveil new cell types and function with high-parameter protein detection
CyTOF2 Mass cytometry system Unveil new cell types and function with high-parameter protein detection DISCOVER MORE. IMAGINE MORE. MASS CYTOMETRY. THE FUTURE OF CYTOMETRY TODAY. Mass cytometry resolves
More informationMAB Solut. MABSolys Génopole Campus 1 5 rue Henri Desbruères 91030 Evry Cedex. www.mabsolut.com. is involved at each stage of your project
Mabsolus-2015-UK:Mise en page 1 03/07/15 14:13 Page1 Services provider Department of MABSolys from conception to validation MAB Solut is involved at each stage of your project Creation of antibodies Production
More informationCOMMITTEE FOR PROPRIETARY MEDICINAL PRODUCTS (CPMP) NOTE FOR GUIDANCE ON THE PRE-CLINICAL EVALUATION OF ANTICANCER MEDICINAL PRODUCTS
The European Agency for the Evaluation of Medicinal Products Human Medicines Evaluation Unit London, 23 July 1998 COMMITTEE FOR PROPRIETARY MEDICINAL PRODUCTS (CPMP) NOTE FOR GUIDANCE ON THE PRE-CLINICAL
More informationAPPLICATION INFORMATION
DRAFT: Rev. D A-2045A APPLICATION INFORMATION Flow Cytometry 3-COLOR COMPENSATION Raquel Cabana,* Mark Cheetham, Jay Enten, Yong Song, Michael Thomas,* and Brendan S. Yee Beckman Coulter, Inc., Miami FL
More informationENDORSED BY THE GOVERNANCE COMMITTEE
Guideline for the Preparation or Manipulation of Monoclonal Antibodies (MABs) and related compounds such as Fusion Proteins, used in the Treatment of Cancer Date Approved by Network Governance July 2012
More informationBiology 3A Laboratory MITOSIS Asexual Reproduction
Biology 3A Laboratory MITOSIS Asexual Reproduction OBJECTIVE To study the cell cycle and understand how, when and why cells divide. To study and identify the major stages of cell division. To relate the
More informationNotes on titering antibodies
Home Events This Week Bulletin Boards Lab Personnel Lab Policies Protocols MolBio Tetramers Proteins Flow Cytometry Immunoassays Instruments Computers Lab Publications Scientiific Literature Science on
More informationSupplementary Information
Supplementary Information Table of Contents Figure S1 1 H NMR spectrum for chromomycin A2 (1) in CDCl3. Figure S2 1 H NMR spectrum expansion for chromomycin A2 (1) in CDCl3. Figure S3 HR-ESI-MS spectrum
More informationPaclitaxel-Loaded Expansile Nanoparticles Enhance Chemotheraputic Drug-delivery in Mesothelioma 3D Multicellular Spheroids
Paclitaxel-Loaded Expansile Nanoparticles Enhance Chemotheraputic Drug-delivery in Mesothelioma 3D Multicellular Spheroids Hongyi Lei 1, Rong Liu 1, Aaron Colby 2, Mark W. Grinstaff 2, Yolonda L. Colson
More informationSupplemental Information. McBrayer et al. Supplemental Data
1 Supplemental Information McBrayer et al. Supplemental Data 2 Figure S1. Glucose consumption rates of MM cell lines exceed that of normal PBMC. (A) Normal PBMC isolated from three healthy donors were
More information2.1.2 Characterization of antiviral effect of cytokine expression on HBV replication in transduced mouse hepatocytes line
i 1 INTRODUCTION 1.1 Human Hepatitis B virus (HBV) 1 1.1.1 Pathogenesis of Hepatitis B 1 1.1.2 Genome organization of HBV 3 1.1.3 Structure of HBV virion 5 1.1.4 HBV life cycle 5 1.1.5 Experimental models
More informationIncorporation of a Novel 3D Cell Culture System to Perform in vitro Cytotoxicity Analyses using Human Primary Hepatocytes
A p p l i c a t i o n N o t e Incorporation of a Novel 3D Cell Culture System to Perform in vitro Cytotoxicity Analyses using Human Primary Hepatocytes Brad Larson, Senior Scientist, Applications Department,
More informationAbstract. Bone marrow-level oxygen tension enables enhanced and sustained growth of 3 new pediatric acute lymphoblastic leukemia cell lines
Abstract Bone marrow-level oxygen tension enables enhanced and sustained growth of 3 new pediatric acute lymphoblastic leukemia cell lines Michael A. Sheard, Min Kang, Daniel Cabral, Joanne Lee, Lilia
More informationQuick Guide to AlphaScreen SureFire Assay Optimization
PPLITION NOT lpha Technology Quick uide to lphascreen Sureire ssay Optimization Introduction Screening kinase activities in cell-based assays offers advantages over the more traditional biochemical approach
More informationFCAP Array v3.0 Software: A New Tool to Analyze BD Cytometric Bead Array (CBA) Data. Monisha Sundarrajan, PhD BD Biosciences
FCAP Array v3.0 Software: A New Tool to Analyze BD Cytometric Bead Array (CBA) Data Monisha Sundarrajan, PhD BD Biosciences 23 13795 00 Agenda Introduction Overview of BD CBA bead based immunoassays FCAP
More informationMicroarray Technology
Microarrays And Functional Genomics CPSC265 Matt Hudson Microarray Technology Relatively young technology Usually used like a Northern blot can determine the amount of mrna for a particular gene Except
More informationAnn.wellhouse@TouchStoneScience.net 1. Enzyme Function
Ann.wellhouse@TouchStoneScience.net 1 Enzyme Function National Science Standards Science as Inquiry: Content Standard A: As a result of activities in grades 9-12, all students should develop: Abilities
More informationApplication Information Bulletin: Set-Up of the CytoFLEX Set-Up of the CytoFLEX* for Extracellular Vesicle Measurement
Application Information Bulletin: Set-Up of the CytoFLEX Set-Up of the CytoFLEX* for Extracellular Vesicle Measurement Andreas Spittler, MD, Associate Professor for Pathophysiology, Medical University
More informationEDF Extended Depth of Field
EDF Extended Depth of Field An upgrade for the ImageStream system Think outside the dot. Break the classic depth of field barrier Improve precision Enhance discrimination Simplify analysis Increase resolution
More informationFlowSight. Flow cytometry with vision
FlowSight Flow cytometry with vision Flow cytometry with vision Introducing FlowSight Capable: Sensitive and flexible for every need Intuitive: Easy-to-use, with imagery for every cell Affordable: Designed
More informationCOURSE TITLE COURSE DESCRIPTION
COURSE TITLE COURSE DESCRIPTION CH-00X CHEMISTRY EXIT INTERVIEW All graduating students are required to meet with their department chairperson/program director to finalize requirements for degree completion.
More informationCytoSelect LDH Cytotoxicity Assay Kit
Product Manual CytoSelect LDH Cytotoxicity Assay Kit Catalog Number CBA-241 960 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction The measurement and monitoring of cell cytotoxicity
More informationCancer SBL101. James Gomes School of Biological Sciences Indian Institute of Technology Delhi
Cancer SBL101 James Gomes School of Biological Sciences Indian Institute of Technology Delhi All Figures in this Lecture are taken from 1. Molecular biology of the cell / Bruce Alberts et al., 5th ed.
More informationScience is hard. Flow cytometry should be easy.
Science is hard. Flow cytometry should be easy. TABLE OF CONTENTS 1 INTRODUCTION TO BD ACCURI C6 SOFTWARE... 1 1.1 Starting BD Accuri C6 Software... 1 1.2 BD Accuri C6 Software Workspace... 2 1.3 Opening
More informationFuture Directions in Clinical Research. Karen Kelly, MD Associate Director for Clinical Research UC Davis Cancer Center
Future Directions in Clinical Research Karen Kelly, MD Associate Director for Clinical Research UC Davis Cancer Center Outline 1. Status of Cancer Treatment 2. Overview of Clinical Research at UCDCC 3.
More informationMulticolor Flow Cytometry: Setup and Optimization on the BD Accuri C6 Flow Cytometer
Multicolor Flow Cytometry: Setup and Optimization on the BD Accuri C6 Flow Cytometer Presented by Clare Rogers, MS Senior Marketing Applications Specialist BD Biosciences 23-13660-00 Webinar Overview Multicolor
More informationWhat is Cancer? Cancer is a genetic disease: Cancer typically involves a change in gene expression/function:
Cancer is a genetic disease: Inherited cancer Sporadic cancer What is Cancer? Cancer typically involves a change in gene expression/function: Qualitative change Quantitative change Any cancer causing genetic
More informationStandard Operating Procedure
1.0 Purpose: 1.1 The characterisation of of main leukocyte subsets in peripheral blood cells from mice by flow cytometry. Reliable values of frequencies of leukocyte clusters are very much dependent on
More informationStandardization, Calibration and Quality Control
Standardization, Calibration and Quality Control Ian Storie Flow cytometry has become an essential tool in the research and clinical diagnostic laboratory. The range of available flow-based diagnostic
More informationThe bleeding edge: emerging issues in biobankingand cryopreservation Allison Hubel, PhD University of Minnesota Preservation as a platform technology Food Core scientific principals Mechanisms of degradation
More informationFuture Oncology: Technology, Products, Market and Service Opportunities
Brochure More information from http://www.researchandmarkets.com/reports/296370/ Future Oncology: Technology, Products, Market and Service Opportunities Description: Future Oncology is an analytical newsletter
More informationLaboratory 5: Properties of Enzymes
Laboratory 5: Properties of Enzymes Technical Objectives 1. Accurately measure and transfer solutions with pipettes 2. Use a Spectrophotometer to study enzyme action. 3. Properly graph a set of data. Knowledge
More informationNo-wash, no-lyse detection of leukocytes in human whole blood on the Attune NxT Flow Cytometer
APPLICATION NOTE Attune NxT Flow Cytometer No-wash, no-lyse detection of leukocytes in human whole blood on the Attune NxT Flow Cytometer Introduction Standard methods for isolating and detecting leukocytes
More informationOn the origin of giant multinuclear Reed-Sternberg cells and the role of CD4 T cells in Hodgkin lymphoma
On the origin of giant multinuclear Reed-Sternberg cells and the role of CD4 T cells in Hodgkin lymphoma Uber die Entstehung von multinuklearen Reed-Sternberg Riesenzellen und die Rolle von CD4 T-Zellen
More informationEdU Flow Cytometry Kit. User Manual
User Manual Ordering information: (for detailed kit content see Table 2) EdU Flow Cytometry Kits for 50 assays: Product number EdU Used fluorescent dye BCK-FC488-50 10 mg 6-FAM Azide BCK-FC555-50 10 mg
More informationLEUKEMIA LYMPHOMA MYELOMA Advances in Clinical Trials
LEUKEMIA LYMPHOMA MYELOMA Advances in Clinical Trials OUR FOCUS ABOUT emerging treatments Presentation for: Judith E. Karp, MD Advancements for Acute Myelogenous Leukemia Supported by an unrestricted educational
More informationCell Biology Questions and Learning Objectives
Cell Biology Questions and Learning Objectives (with hypothetical learning materials that might populate the objective) The topics and central questions listed here are typical for an introductory undergraduate
More informationWhat Cytometry Can Do for You: The Pros and Cons of Image and Flow Cytometry Webinar 31 October 2012
What Cytometry Can Do for You: The Pros and Cons of Image and Flow Cytometry Webinar 31 October 2012 [0:00:10] Slide 1 Hello everyone and welcome to this Science/AAAS webinar. I m Sean Sanders, editor
More informationThe Huntington Library, Art Collections, and Botanical Gardens
The Huntington Library, Art Collections, and Botanical Gardens Rooting for Mitosis Overview Students will fix, stain, and make slides of onion root tips. These slides will be examined for the presence
More informationChapter 8. Summary and Perspectives
Chapter 8 Summary and Perspectives 131 Chapter 8 Summary Overexpression of the multidrug resistance protein MRP1 confer multidrug resistance (MDR) to cancer cells. The contents of this thesis describe
More informationImmunophenotyping peripheral blood cells
IMMUNOPHENOTYPING Attune Accoustic Focusing Cytometer Immunophenotyping peripheral blood cells A no-lyse, no-wash, no cell loss method for immunophenotyping nucleated peripheral blood cells using the Attune
More informationGuided Notes: Chapter 9 Cellular Reproduction
Guided Notes: Cellular Reproduction When do cells divide? Cells grow and function normally until they become too. Cell size is because increases faster than This means that there is not enough area on
More informationCyFlow SL. Microbiology. Detection and Analysis of Microorganisms and Small Particles
CyFlow SL Microbiology Detection and Analysis of Microorganisms and Small Particles 01 COMPANY Flow Cytometry made in Germany New sophisticated applications and increasing requirements for reliable results
More informationgreen B 1 ) into a single unit to model the substrate in this reaction. enzyme
Teacher Key Objectives You will use the model pieces in the kit to: Simulate enzymatic actions. Explain enzymatic specificity. Investigate two types of enzyme inhibitors used in regulating enzymatic activity.
More informationRoutine Assessment of Cancer Cell Cytotoxicity in a Novel Three Dimensional Culture Assay. Introduction
Routine Assessment of Cancer Cell Cytotoxicity in a Novel Three Dimensional Culture Assay Application Note Introduction Cancer is a leading cause of death worldwide, accounting for around 7.6 million deaths
More information岑 祥 股 份 有 限 公 司 技 術 專 員 費 軫 尹 20100803
技 術 專 員 費 軫 尹 20100803 Overview of presentation Basic Biology of RNA interference Application of sirna for gene function? How to study mirna? How to deliver sirna and mirna? New prospects on RNAi research
More informationCellulär och molekylär respons på låga doser av joniserande strålning
Cellulär och molekylär respons på låga doser av joniserande strålning Bo Stenerlöw Biomedicinsk strålningsvetenskap Rudbecklaboratoriet Uppsala universitet Bo Stenerlöw Biomedical Radiation Sciences Uppsala
More informationSupplementary Materials for
www.sciencesignaling.org/cgi/content/full/7/339/ra80/dc1 Supplementary Materials for Manipulation of receptor oligomerization as a strategy to inhibit signaling by TNF superfamily members Julia T. Warren,
More informationApplications of comprehensive clinical genomic analysis in solid tumors: obstacles and opportunities
Applications of comprehensive clinical genomic analysis in solid tumors: obstacles and opportunities Vincent A. Miller, M.D. Foundation Medicine, Inc. AACR Annual Meeting 2012 Current Concepts session
More informationRoutine Assessment of Cancer Cell Cytotoxicity in a Novel Three Dimensional Culture Assay. Introduction
Routine Assessment of Cancer Cell Cytotoxicity in a Novel Three Dimensional Culture Assay Application Note 2 Introduction Cancer is a leading cause of death worldwide, accounting for around 7.6 million
More information