In vitro functional study of Cardiomyocyte
|
|
|
- Rodger Colin Joseph
- 5 years ago
- Views:
Transcription
1 In vitro functional study of Cardiomyocyte Gwang Hyeon Eom Department of Pharmacology and Medical Research Center for Gene Regulation Chonnam National University Medical School, Gwangju, South Korea
2 Heart and cardiomyocyte or Sarcomeric actin Sarcomeric alphaactinin (Zlines) Nucleus A: Adult Rat Cardiac Myocyte (nucleus not shown) B: Neonatal Rat Cardiac Myocyte
3 Characteristics of cardiomyocytes 1. Involuntarystriated muscle 2. Can contain one, two, or very rarely three or four cell nuclei 3. Oxygen and nutrients are supplied not by blood in the cardiac chamber directly but by coronary arteries. 4. Cardiac muscle requires extracellular calcium ions for contraction to occur 5. Until recently, it was commonly believed that cardiac muscle cells could not be regenerated. However, a study reported in the April 3, 2009 issue of Science contradicts that belief. Olaf Bergmann and his colleagues at the Karolinska Institute in Stockholm tested samples of heart muscle from people born before 1955 when nuclear bomb testing caused elevated levels of radioactive carbon 14 in the Earth's atmosphere. They found that samples from people born before 1955 did have elevated carbon 14 in their heart muscle cell DNA, indicating that the cells had divided after the person's birth.
4 Sources of cardiac cells 1. Primary cultured a. Human adult from biopsy b. Mouse neonate, adult c. Rat neonate, adult
5 Sources of cardiac cells 2. Immortalized cell line A. AC16 human 1 2 Adult human ventricle origin Fused with SV40, having large T antigen Davidson MM et al., J Mol Cell Cardiol Jul;39(1):133-47
6 Sources of cardiac cells 2. Immortalized cell line B. HL1 mouse 1 2 Retains contractile phenotype supplemented with 2 mm Lglutamine 0.1 mm norepinephrine
7 Sources of cardiac cells 2. Immortalized cell line C. H9c2 rat 1 2 Avoid to be confluent Can be differentiated to skeletal myocyte by serum deprivation or by retinoic acid
8 Neonatal rat ventricular cardiomyocytes (NRVCMs) Materials 1. SD Rat pubs (PN 1d ~ 2d) 2. Ads buffer 120 mm NaCl, 6 mm glucose, 8 mm NaH 2 PO 4, 5 mm KCl, 0.8 mm MgSO 4, 20 mm HEPES, ph Collagenase (type II) 0.5 mg/ml 4. Pancreatin 0.6 mg/ml 5. Percoll 6. Gelatin or collagen
9 Neonatal rat ventricular cardiomyocytes (NRVCMs) Methods 1. Isolate heart from rat pubs 2. Remove appendages (use only ventricle) 3. Mince the ventricles 4. Enzyme digestion (Collagenase/Pancreatin/Ads buffer) 5. Percoll gradient or preplating without coating 6. Coat the culture plate with collagen or gelatin 7. Seed the NRVCMs 2x10 6 / Check the NRVCMs beating
10 Adult cardiac myocytes (Mouse) Methods 1. 10X Digestion buffer (DB) NaCl KCl Pyruvic acid HEPES MgCl 2 NaH 2 PO 4 Dextrose Adjust ph to mM 5mM 3nM 25mM 0.5mM 0.33mM 22mM
11 Adult cardiac myocytes (Mouse) Methods 2. Digestion Buffer A 50mL 1x DB with 75ul EGTA(100mM EGTA) 3. Digestion Buffer B 25mL 1x DB with 1.25uL CaCl 2 (1M) 1mg Protease, 25mg Collagenase 4. Collection buffer (37 o C) 2.5mL 1X DB with 1.25ul CaCl 2 (1M) 0.5mg Protease, 15mg Collagenase, 125mg BSA 5. Neutralization Wash Buffer 25mL 1X DB with 6.25ul CaCl 2 (1M) 250mg BSA
12 Adult cardiac myocytes (Mouse) Methods 1. Isolate heart from mouse and keep cold with Buffer A 2. Briefly rinse heart with ice cold Buffer A 3. Perfuse the heart with 37 o C fluid (Buffer A) Langendorff system 4. Switch to the Buffer B from A after blood is washed from the heart 5. Cut the ventricles off with scissors and place into Collection Buffer(CB) 6. Gently cut the ventricle into small pieces while placing into the CB 7. Let the larger tissue pieces sink to the bottom 8. Remove and save the supernatant 9. Let gravity pull a pellet from the supernatant for 15 minutes at RT 10. Remove the supernatant and wash pellet by adding the Neutralization buffer. Flynn JM et al., J Vis Exp Dec 28;(58):e3302. doi: /3302.
13 Limitations of NRVCMs or H9c2 1. NRVCMs a. Risk of bacterial contamination b. Yield c. Subculture d. Extremely low transfection efficiency viral delivery system 2. H9c2 a. Myoblast cell b. Lack of cardiac phenotype
14 Usage of NRVCMs or H9c2 1. H9c2 a. General cell works except chemical treatment 2. NRVCMs a. ICC b. Cell size measurement c. Promoter assay d. cell works, especially chemical study when using viral delivery system, NRVCMs is the cell of choice.
15 Commercially available primary cells (BioTrader, Inc) (Abba BioScience)
16 Examples
17 Hypertrophic stimuli phosphorylate Hdac2 S394 IB:a-Hdac2 thdac2 IB:a-Hdac2 thdac2
18 Phosphorylation of HDAC2 S394 is required for the development of cardiac hypertrophy
19 Casein Kinase 2 phosphorylates Hdac2 S394
20 Casein Kinase 2 phosphorylates Hdac2 S394
21 CK2 is activated in response to hypertrophic stimuli
22 Hdac2 S394A blunts the hypertrophic phenotypes of CK2 1
23 Hdac2 S394A blunts the hypertrophic phenotypes of CK2 1 Eom et al., Circulation 123(21): , 2011
24 Crosstalk between Hdac2 and Class IIa HDACs McKinsey TA and Olson EN. Trends Genet, 20(4):206-13, 2004 Kee and Eom et al., Circ Res, 103(11): , 2008
25 Hdac2 is acetylated in the development of cardiac hypertrophy A IP: Acetyl Lysine B IP: Acetyl Lysine IB: Hdac2 AcHdac2 (output) Hdac2 (Input) IB: Hdac2 AcHdac2 (Output) Hdac2 (Input) ET1: ISP: Cardiomyocyte lysates Heart lysates
26 Hdac2 acetylation is reduced by Garcinol but increased by TSA B 1.6 A IP: IB: Hdac2 Hdac2 wt : Garcinol : TSA : Acetyl Lysine Cardiomyocyte lysates AcHdac2 (output) Hdac2 (Input) Garcinol : HAT(histone acetyl transferase) inhibitor TSA : HDAC(histone deacetylase) inhibitor Hdac2 activity (relative change) Hdac2wt Garcinol TSA l l l l
27 Which residue(s) is(are) the acetylation target(s)?
28 Acetylation target of Hdac2 is located on the A part A part : 249 a.a B part : 239 a.a HDAC domain Hdac2 full Hdac2 A Hdac2 B Aceylated Hdac2 Aceylated Hdac2 Input IB: V5 IP: AcK IB: V5 IP: AcK IB: V5
29 K75 is the target of acetylation IP: IgG Acetyl Lysine IB: V5 Acetyl Hdac2 IgG heavy chain Input IB: V5 Hdac2 wt : Hdac2 K75R : Hdac2 K90R : A IP: IgG Acetyl Lysine IB: Hdac2 AcHdac2 Total Hdac2 Hdac2 wt : Hdac2 K75R :
30 Which HATase?/Which HDAC?
31 P300/CBPassociated factor binds to Hdac2 for actetylation C IP: IgG Flag(pCAF) IB: V5(Hdac2) Output IB: Flag(pCAF) Input D IP: IgG Acetyl Lysine IB: V5(Hdac2) Output IB: pcaf Input IB: GAPDH
32 Class IIa HDAC bind to Hdac2 A IP: IB: V5 (Hdac2) Flag (Hdac5) V5 (Hdac2) Flag(Hdac5) Output Input C IB: Hdac2 Hdac9 WCE IgG Hdac9 :IP Hdac2V5 : FlagHdac5 : B IP: IB: Flag (Hdac5) Flag (Hdac5) IgG Hdac2 Output Input D IB: V5 (Hdac9) Hdac2 WCE IP: IgG Hdac2 Hdac2
33 Hdac5 deacetylase Hdac2, which is completely reversed by nonspecific HDAC inhibitor IP: IgG Acetyl Lysine V5 (Hdac2) Output Flag (pcaf) Input Flag (Hdac5) Hdac2V5 FlagpCAF Hdac5flag TSA
34 How Acetylation affect to enzymatic activity of Hdac2??
35 Acetylation of Hdac2 affects phosphorylation status A B IP: Acetyl Lysine HDAC activity (fold change, arbitary units) IB: V5 (Hdac2) Hdac2 wt Hdac2 K75R TSA C IP: IB: p394 Hdac2 V5(Hdac2) AcHdac2 Total Hdac2 Hdac2 wt Hdac2 K75R pcaf V5 (Hdac2) Hdac2 wt Hdac2 K75R TSA
36 Acetylation of Hdac2 affects phosphorylation status A IP: IgG Acetyl Lysine V5 (Hdac2) Output Flag (pcaf) Input Flag (Hdac5) Hdac2V5 FlagpCAF Hdac5flag TSA B Hdac5Flag : TSA : p394 Hdac2 Hdac2 Eom et al., in prepration Flag (Hdac5)
37 Working hypothesis Kee and Eom et al., Circ Res, 103(11): , 2008 CK2 1 Quiet state Activated state P Class IIa HDAC inactivated state Hdac2 Class IIa HDAC pcaf CK2 1 P Hdac2 Ac pcaf Hdac2???
38 Thank you for your attention!!
CANINE CARDIAC MYOCYTE ISOLATION PROTOCOL November 2000
CANINE CARDIAC MYOCYTE ISOLATION PROTOCOL November 2000 I. Preparation for cell isolation: A. Make sure the following items are autoclaved: 1. 1-8X8 baking dish 6. 3-1 liter bottles 2. 2-500 ml beaker
Supporting Information
Supporting Information Muraski et al. 10.1073/pnas.0709135105 SI Text Generation of Transgenic Animals. Pim-WT and Pim-DN cdnas were subcloned NheI/SmaI from pegfp-c1 Pim-1 and pegfp-c1 Pim-DN plasmids
2. Langendorff Heart
2. Langendorff Heart 2.1. Principle Langendorff heart is one type of isolated perfused heart which is widely used for biochemical, physiological, morphological and pharmacological researches. It provides
SUPPLEMENTAL MATERIAL. Supplementary Methods
SUPPLEMENTAL MATERIAL Supplementary Methods Culture of cardiomyocytes, fibroblasts and cardiac microvascular endothelial cells The isolation and culturing of neonatal rat ventricular cardiomyocytes was
Supplementary Figure 1. MAT IIα is Acetylated at Lysine 81.
IP: Flag a Mascot PTM Modified Mass Error Position Gene Names Score Score Sequence m/z [ppm] 81 MAT2A;AMS2;MATA2 35.6 137.28 _AAVDYQK(ac)VVR_ 595.83-2.28 b Pre-immu After-immu Flag- WT K81R WT K81R / Flag
Glucose Cl NaCl K 2.5
Brains were removed and immersed in ice- cold solution containing the following (in mm): 87 NaCl, 2.5 KCl, 1.25NaH2PO4, 7MgSO4, 0.5 CaCl2, 25 NaHCO3, 25 glucose, and 75 sucrose, ph 7.4, 315 mosm. Parasagittal
FOCUS SubCell. For the Enrichment of Subcellular Fractions. (Cat. # ) think proteins! think G-Biosciences
169PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name FOCUS SubCell For the Enrichment of Subcellular Fractions (Cat. # 786 260) think
condition. Left panel, the HCT-116 cells were lysed with RIPA buffer containing 0.1%
FIGURE LEGENDS Supplementary Fig 1 (A) sumoylation pattern detected under denaturing condition. Left panel, the HCT-116 cells were lysed with RIPA buffer containing 0.1% SDS in the presence and absence
ab Histone Deacetylase (HDAC) Activity Assay Kit (Fluorometric)
ab156064 Histone Deacetylase (HDAC) Activity Assay Kit (Fluorometric) Instructions for Use For the quantitative measurement of Histone Deacetylase activity in cell lysates This product is for research
5.1. Isolation and Culture of Adult Ventricular Cardiomyocytes. Introduction
557 Isolation and Culture of Adult Ventricular Cardiomyocytes Klaus-Dieter Schlüter and Hans Michael Piper Introduction The current understanding of the physiological and pathophysiological behaviour of
Kidney. Heart. Lung. Sirt1. Gapdh. Mouse IgG DAPI. Rabbit IgG DAPI
a e Na V 1.5 Ad-LacZ Ad- 110KD b Scn5a/ (relative to Ad-LacZ) f 150 100 50 0 p = 0.65 Ad-LacZ Ad- c Heart Lung Kidney Spleen 110KD d fl/fl c -/- DAPI 20 µm Na v 1.5 250KD fl/fl Rabbit IgG DAPI fl/fl Mouse
Gallic acid prevents isoproterenol-induced cardiac hypertrophy and fibrosis through regulation of JNK2 signaling and Smad3 binding activity
Gallic acid prevents isoproterenol-induced cardiac hypertrophy and fibrosis through regulation of JNK2 signaling and Smad3 binding activity Yuhee Ryu 1,+, Li Jin 1,2+, Hae Jin Kee 1,, Zhe Hao Piao 3, Jae
2-Deoxyglucose Assay Kit (Colorimetric)
2-Deoxyglucose Assay Kit (Colorimetric) Catalog Number KA3753 100 assays Version: 01 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 General Information...
SUPPLEMENTARY INFORMATION
Supplementary Figures Supplementary Figure S1. Binding of full-length OGT and deletion mutants to PIP strips (Echelon Biosciences). Supplementary Figure S2. Binding of the OGT (919-1036) fragments with
Acta Physiologica Sinica
, 1999 4, 51 (2), 187 192 187 Acta Physiologica Sinica 3 1998204222 1998206203 3 (No139500052) 3 3, 221002 3 3 3 3 3 (, 200031) ( Ito), 28 d (H28, 6 h/ d), Ito (16118 4161 6132 1135 pa/ pf, P < 0105),
Minute TM Plasma Membrane Protein Isolation and Cell Fractionation Kit User Manual (v5)
Minute TM Plasma Membrane Protein Isolation and Cell Fractionation Kit Catalog number: SM-005 Description Minute TM plasma membrane (PM) protein isolation kit is a novel and patented native PM protein
Trident Membrane Protein Extraction Kit
Cat. No. Size Shelf life GTX16373 5/ 20 tests 12 months at the appropriate storage temperatures (see below) Contents Component Storage Amount for 5 tests Amount for 20 tests Buffer A -20 o C 2.5 ml 10
Mouse primary keratinocytes preparation
Mouse primary keratinocytes preparation 1. Fill a 150 X 25 mm petri dish with ice. Put newborn mice (2 3 days old) in the petri dish and insert it in an ice bucket. Leave the mice in the ice bucket for
Modulating Glucose Uptake in Skeletal Myotubes: Insulin Induction with Bioluminescent Glucose Uptake Analysis
icell Skeletal Myoblasts Application Protocol Modulating Glucose Uptake in Skeletal Myotubes: Insulin Induction with Bioluminescent Glucose Uptake Analysis Introduction The skeletal muscle is one of the
SUPPLEMENTARY INFORMATION
a c e doi:10.1038/nature10407 b d f Supplementary Figure 1. SERCA2a complex analysis. (a) Two-dimensional SDS-PAGE gels of SERCA2a complexes. A silver-stained SDSPAGE gel is shown, which reveals a 12 kda
Total Histone H3 Acetylation Detection Fast Kit (Colorimetric)
Total Histone H3 Acetylation Detection Fast Kit (Colorimetric) Catalog Number KA1538 48 assays Version: 02 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use...
HDAC Fluorometric Activity Assay Kit
HDAC Fluorometric Activity Assay Kit Item. 10011563 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
EpiQuik Total Histone H3 Acetylation Detection Fast Kit (Colorimetric)
EpiQuik Total Histone H3 Acetylation Detection Fast Kit (Colorimetric) Base Catalog # PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik Total Histone H3 Acetylation Detection Fast Kit (Colorimetric)
Western Immunoblotting Preparation of Samples:
Western Immunoblotting Preparation of Samples: Total Protein Extraction from Culture Cells: Take off the medium Wash culture with 1 x PBS 1 ml hot Cell-lysis Solution into T75 flask Scrap out the cells
THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY
Page: 1 of 5 1. Risk Assessment: This Risk Assessment is to be used as a general guide and as such, cannot accommodate all the varying factors that may be encountered when using this procedure. Therefore,
ANSC/FSTC 607 Biochemistry and Physiology of Muscle as a Food PRIMARY, SECONDARY, AND TERTIARY MYOTUBES
ANSC/FSTC 607 Biochemistry and Physiology of Muscle as a Food PRIMARY, SECONDARY, AND TERTIARY MYOTUBES I. Satellite Cells A. Proliferative, myoblastic cells that lie in invaginations in the sarcolemma
PREPARATION OF IF- ENRICHED CYTOSKELETAL PROTEINS
TMM,5-2011 PREPARATION OF IF- ENRICHED CYTOSKELETAL PROTEINS Ice-cold means cooled in ice water. In order to prevent proteolysis, make sure to perform all steps on ice. Pre-cool glass homogenizers, buffers
2-Deoxyglucose (2DG) Uptake Measurement kit
Document#:K2DG13516E For research use only. Not for clinical diagnosis. Catalog No. CSR-OKP-PMG-K1E 2-Deoxyglucose (2DG) Uptake Measurement kit Introduction Measurement of 2-deoxyglucose (2DG) uptake in
Human TRPC6 Ion Channel Cell Line
TECHNICAL DATA SHEET ValiScreen Ion Channel Cell Line Caution: For Laboratory Use. A research product for research purposes only Human TRPC6 Ion Channel Cell Line Product No.: AX-012-C Lot No.: 512-548-A
Hepatitis B Antiviral Drug Development Multi-Marker Screening Assay
Hepatitis B Antiviral Drug Development Multi-Marker Screening Assay Background ImQuest BioSciences has developed and qualified a single-plate method to expedite the screening of antiviral agents against
BILAYER CHANNEL RECONSTITUTION
(1) 1% Agar Salt Bridge 1.0 g Agar 3.75g KCl in 100ml distilled water, store at 4 o C. BILAYER CHANNEL RECONSTITUTION (2) Cs solution: (Cesium Methanesulfonate) 1) 50 mm Cs + solution 0.209 MOPS, 10mM
-adrenergic receptor subtypes blockade on the rat myocardium inotropy
Research Article The effect of -adrenergic receptor subtypes blockade on the rat myocardium inotropy Insaf Ilkhamovich Khabibrakhmanov*, Nafisa Ilgizovna Ziшatdinova, Timur Lvovich Zefirov ABSTRACT Aim:
EPIGENTEK. EpiQuik Global Acetyl Histone H3K27 Quantification Kit (Colorimetric) Base Catalog # P-4059 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE
EpiQuik Global Acetyl Histone H3K27 Quantification Kit (Colorimetric) Base Catalog # P-4059 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik Global Acetyl Histone H3K27 Quantification Kit (Colorimetric)
SUPPLEMENTARY INFORMATION
doi:10.1038/nature12652 Supplementary Figure 1. PRDM16 interacts with endogenous EHMT1 in brown adipocytes. Immunoprecipitation of PRDM16 complex by flag antibody (M2) followed by Western blot analysis
SUPPLEMENTARY INFORMATION
doi:10.1038/nature11700 Figure 1: RIP3 as a potential Sirt2 interacting protein. Transfected Flag-tagged Sirt2 was immunoprecipitated from cells and eluted from the Sepharose beads using Flag peptide.
Antibodies: LB1 buffer For 50 ml For 10ml For 30 ml Final 1 M HEPES, ph 2.5 ml 0.5 ml 1.5 ml 50mM. 5 M NaCl 1.4 ml 280 µl 0.
Experiment: Date: Tissue: Purpose: ChIP-Seq Antibodies: 11x cross-link buffer: Regent Stock Solution Final Vol for 10 ml of 11xstock concentration 5 M NaCl 0.1M 0.2 ml 0.5 M EDTA 1 mm 20 ul 0.5 M EGTA,
BIL 256 Cell and Molecular Biology Lab Spring, Tissue-Specific Isoenzymes
BIL 256 Cell and Molecular Biology Lab Spring, 2007 Background Information Tissue-Specific Isoenzymes A. BIOCHEMISTRY The basic pattern of glucose oxidation is outlined in Figure 3-1. Glucose is split
For the 5 GATC-overhang two-oligo adaptors set up the following reactions in 96-well plate format:
Supplementary Protocol 1. Adaptor preparation: For the 5 GATC-overhang two-oligo adaptors set up the following reactions in 96-well plate format: Per reaction X96 10X NEBuffer 2 10 µl 10 µl x 96 5 -GATC
Product Use HPSC-CC are for research use only. It is not approved for human or animal use, or for application in in vitro diagnostic procedures.
HPSC-derived Cardiomyocyte Cells (HPSC-CC) Catalog #6240 Cell Specification Human primary cardiomyocytes and cardiac tissue are superior modeling systems for heart disease studies, drug discovery and toxicity
7.06 Cell Biology EXAM #3 April 24, 2003
7.06 Spring 2003 Exam 3 Name 1 of 8 7.06 Cell Biology EXAM #3 April 24, 2003 This is an open book exam, and you are allowed access to books and notes. Please write your answers to the questions in the
Glucose Uptake Colorimetric Assay Kit
ab136955 Glucose Uptake Colorimetric Assay Kit Instructions for Use For the sensitive and accurate measurement of Glucose uptake in various samples This product is for research use only and is not intended
Supplemental Experimental Procedures
Cell Stem Cell, Volume 2 Supplemental Data A Temporal Switch from Notch to Wnt Signaling in Muscle Stem Cells Is Necessary for Normal Adult Myogenesis Andrew S. Brack, Irina M. Conboy, Michael J. Conboy,
PHYSIOLOGY MeQ'S (Morgan) All the following statements related to blood volume are correct except for: 5 A. Blood volume is about 5 litres. B.
PHYSIOLOGY MeQ'S (Morgan) Chapter 5 All the following statements related to capillary Starling's forces are correct except for: 1 A. Hydrostatic pressure at arterial end is greater than at venous end.
Special circulations, Coronary, Pulmonary. Faisal I. Mohammed, MD,PhD
Special circulations, Coronary, Pulmonary Faisal I. Mohammed, MD,PhD 1 Objectives Describe the control of blood flow to different circulations (Skeletal muscles, pulmonary and coronary) Point out special
Data Sheet. Fluorogenic HDAC 8 Assay Kit Catalog #: 50068
Data Sheet Fluorogenic HDAC 8 Assay Kit Catalog #: 50068 DESCRIPTION: The Fluorogenic HDAC 8 Assay Kit is a complete assay system designed to measure histone deacetylase (HDAC) 8 activity for screening
Application Note. Introduction
Simultaneously Measuring Oxidation of Exogenous and Endogenous Fatty Acids Using the XF Palmitate-BSA FAO Substrate with the Agilent Seahorse XF Cell Mito Stress Test Application Note Introduction The
InPulse: Development of an ipsc cardiomyocyte (ipsc-cm) platform to assess drug-induced contractility liabilities.
InPulse: Development of an ipsc cardiomyocyte (ipsc-cm) platform to assess drug-induced contractility liabilities. Sponsors: Arun Sridhar, Peter J. Clements, Brian Berridge, Drug induced contractility
Thawing MEFs (Mouse Embryonic Fibroblasts (MEFs)
1 FEEDER CULTURES The function of feeder cultures is to support the undifferentiated growth of hpsc. Typically primary fibroblasts are used for this purpose. We prepare our mouse feeder cells from ICR
Recombinant Protein Expression Retroviral system
Recombinant Protein Expression Retroviral system Viruses Contains genome DNA or RNA Genome encased in a protein coat or capsid. Some viruses have membrane covering protein coat enveloped virus Ø Essential
INSTRUCTIONS Pierce Primary Cardiomyocyte Isolation Kit
INSTRUCTIONS Pierce Primary Cardiomyocyte Isolation Kit 88281 Number Description 88281 Pierce Primary Cardiomyocyte Isolation Kit, contains sufficient reagents to isolate cardiomyocytes from 50 neonatal
Chromatin Immunoprecipitation (ChIPs) Protocol (Mirmira Lab)
Chromatin Immunoprecipitation (ChIPs) Protocol (Mirmira Lab) Updated 12/3/02 Reagents: ChIP sonication Buffer (1% Triton X-100, 0.1% Deoxycholate, 50 mm Tris 8.1, 150 mm NaCl, 5 mm EDTA): 10 ml 10 % Triton
Microanatomy of Muscles. Anatomy & Physiology Class
Microanatomy of Muscles Anatomy & Physiology Class Three Main Muscle Types Objectives: By the end of this presentation you will have the information to: 1. 2. 3. 4. 5. 6. Describe the 3 main types of muscles.
Resident cardiac stem cells: how to find and use them
Resident cardiac stem cells: how to find and use them G. Hasenfuß Cardiology and Pneumology Heart Research Center Göttingen Georg-August-University Göttingen Definition: Stem cell Selfrenewal Stem cell
20X Buffer (Tube1) 96-well microplate (12 strips) 1
PROTOCOL MitoProfile Rapid Microplate Assay Kit for PDH Activity and Quantity (Combines Kit MSP18 & MSP19) 1850 Millrace Drive, Suite 3A Eugene, Oregon 97403 MSP20 Rev.1 DESCRIPTION MitoProfile Rapid Microplate
Lumino Firefly Luciferase Assay
G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Lumino Firefly Luciferase Assay (Cat. # 786 1267, 786 1268) think proteins! think G-Biosciences
Outline. Bio 105: Muscular System. Muscular System. Types of Muscles. Smooth Muscle. Cardiac Muscle 4/6/2016
Outline Bio 105: Muscular System Lecture 11 Chapter 6 Characteristics of muscles 3 types of muscles Functions of muscles Structure of skeletal muscles Mechanics of muscle contraction Energy sources for
Mammalian Tissue Protein Extraction Reagent
Mammalian Tissue Protein Extraction Reagent Catalog number: AR0101 Boster s Mammalian Tissue Protein Extraction Reagent is a ready-to-use Western blot related reagent solution used for efficient extraction
Relaxation responses of aortic rings from salt-loaded high calcium fed rats to potassium chloride, calcium chloride and magnesium sulphate
Pathophysiology 4 (1998) 275 280 Relaxation responses of aortic rings from salt-loaded high calcium fed rats to potassium chloride, calcium chloride and magnesium sulphate B.J. Adegunloye, O.A. Sofola
EXAM II Animal Physiology ZOO 428 Fall 2006
V Eq EXAM II Animal Physiology ZOO 428 Fall 2006 = RT X o. ln( [ zf [ X ) RT p K[K o pna[na o pcl[cl i V = m ln i F pk[k i pna[na i pcl[cl o I = g(v m V eq. ) Q = C m V m Q Driving Force = V m V eq. 10
Mouse sperm extraction:
Mouse sperm extraction: This method of extraction is used for acrosome reaction assays, immunocytochemistry and biochemical assays. Collect two cauda epidydimus from one male, cut them 5 times and place
Impact of hyper-o-glcnacylation on apoptosis and NF-κB activity SUPPLEMENTARY METHODS
SUPPLEMENTARY METHODS 3D culture and cell proliferation- MiaPaCa-2 cell culture in 3D was performed as described previously (1). Briefly, 8-well glass chamber slides were evenly coated with 50 µl/well
The Schedule and the Manual of Basic Techniques for Cell Culture
The Schedule and the Manual of Basic Techniques for Cell Culture 1 Materials Calcium Phosphate Transfection Kit: Invitrogen Cat.No.K2780-01 Falcon tube (Cat No.35-2054:12 x 75 mm, 5 ml tube) Cell: 293
Pluricyte Cardiomyocytes
Pluricyte Cardiomyocytes Manual Version 2.1 / March 2018 Contents 1. Introduction 2 2. Equipment, Materials and Reagents 3 3. Methods 4 3.1 Coating of tissue culture plates 4 3.2 Thawing Pluricyte Cardiomyocytes
ab Glucose Uptake Assay Kit (colorimetric) 1
Version 16 Last updated 10 January 2018 ab136955 Glucose Uptake Assay Kit (Colorimetric) For the measurement of Glucose uptake in a variety of cells. This product is for research use only and is not intended
Product Size Catalog Number. 500,000 proliferating cells. cryopreserved cells that have been thawed and cultured for three days at PromoCell.
Cardiac Myocytes Instruction Manual Product Size Catalog Number Human Cardiac Myocytes (HCM) 500,000 cryopreserved cells 500,000 proliferating cells C-12810 C-12811 Product Description Cardiac muscle,
2013 W. H. Freeman and Company. 12 Signal Transduction
2013 W. H. Freeman and Company 12 Signal Transduction CHAPTER 12 Signal Transduction Key topics: General features of signal transduction Structure and function of G protein coupled receptors Structure
Mammalian Cell PE LB
257PR G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Mammalian Cell PE LB Mammalian Cell Protein Extraction & Lysis Buffer (Cat. # 786 180)
Skeletal muscle in the light of its structure
Mechanism of contraction of Skeletal muscle in the light of its structure By Dr. Mudassar Ali Roomi (MBBS, M. Phil) Muscle Tissue Skeletal Muscle Cardiac Muscle Smooth Muscle Skeletal Muscle Long cylindrical
The Cardiovascular System
Chapter 18 Part A The Cardiovascular System 1/19/16 1 Annie Leibovitz/Contact Press Images Similarities of Cardiac and Skeletal Muscle RMP Ion concentration Deploarization Action Potential Repolarization
Medical Biochemistry and Molecular Biology department
Medical Biochemistry and Molecular Biology department Cardiac Fuels [Sources of energy for the Cardiac muscle] Intended learning outcomes of the lecture: By the end of this lecture you would be able to:-
Glucose Uptake Assay Kit (Fluorometric)
ab136956 Glucose Uptake Assay Kit (Fluorometric) Instructions for Use For the sensitive and accurate measurement of Glucose uptake in various samples This product is for research use only and is not intended
Supplemental Information. T Cells Enhance Autoimmunity by Restraining Regulatory T Cell Responses via an Interleukin-23-Dependent Mechanism
Immunity, Volume 33 Supplemental Information T Cells Enhance Autoimmunity by Restraining Regulatory T Cell Responses via an Interleukin-23-Dependent Mechanism Franziska Petermann, Veit Rothhammer, Malte
Circulation: Chapter 25. Cardiac Output. The Mammalian Heart Fig Right side of the heart
Circulation: Chapter 25 1. Limits of Diffusion A. Small organisms use diffusion B. rapid over small distances 2. Most animals have circulatory systems A. Blood B. Pump (Heart) or propulsive structures
Essential Medium, containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Huvec were cultured in
Supplemental data Methods Cell culture media formulations A-431 and U-87 MG cells were maintained in Dulbecco s Modified Eagle s Medium. FaDu cells were cultured in Eagle's Minimum Essential Medium, containing
AMPK Phosphorylation Assay Kit
AMPK Phosphorylation Assay Kit Catalog Number KA3789 100 assays Version: 02 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use... 3 Background... 3 Principle
Supplemental Material. Methods
1 Supplemental Material Methods Functional Characterization of Isolated Cardiomyocytes (Detailed Methods) Left ventricular myocytes were isolated from adult mice (aged 30-38 weeks) by enzymatic dissociation.
T H E J O U R N A L O F C E L L B I O L O G Y
Supplemental material Jewell et al., http://www.jcb.org/cgi/content/full/jcb.201007176/dc1 T H E J O U R N A L O F C E L L B I O L O G Y Figure S1. IR Munc18c association is independent of IRS-1. (A and
EpiQuik Circulating Acetyl Histone H3K18 ELISA Kit (Colorimetric)
EpiQuik Circulating Acetyl Histone H3K18 ELISA Kit (Colorimetric) Base Catalog # PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses: The EpiQuik Circulating Acetyl Histone H3K18 ELISA Kit (Colorimetric)
CHAPTER 4 IMMUNOLOGICAL TECHNIQUES
CHAPTER 4 IMMUNOLOGICAL TECHNIQUES Nitroblue Tetrazolium Chloride (NBT) Reduction test NBT reduction test was evaluated by employing the method described by Hudson and Hay,1989 based upon principle that
Age-related changes in cardiovascular system. Dr. Rehab Gwada
Age-related changes in cardiovascular system Dr. Rehab Gwada Objectives explain the main structural and functional changes in cardiovascular system associated with normal aging Introduction aging results
Chapter 10 Muscle Tissue and Physiology Chapter Outline
Chapter 10 Muscle Tissue and Physiology Chapter Outline Module 10.1 Overview of muscle tissue (Figures 10.1 10.2) A. Types of Muscle Tissue (Figure 10.1) 1. The three types of cells in muscle tissue are,,
SI Materials and methods
SI Materials and methods Cell culture and transfection. Primary cultures of neonatal cardiac myocytes were prepared as described previously 1. Hearts were removed from rat pups at postnatal day 1 (P1).
EPIGENTEK. EpiQuik Total Histone Extraction Kit. Base Catalog # OP-0006 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE
EpiQuik Total Histone Extraction Kit Base Catalog # PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses: The EpiQuik Total Histone Extraction Kit is suitable for a quick preparation of total histone extracts
INTERACTION DRUG BODY
INTERACTION DRUG BODY What the drug does to the body What the body does to the drug Receptors - intracellular receptors - membrane receptors - Channel receptors - G protein-coupled receptors - Tyrosine-kinase
Calcineurin Cellular Activity Assay Kit, Colorimetric Cat. No
User Protocol 207007 Rev. 27-July-04 JSW Page 1 of 9 Calcineurin Cellular Activity Assay Kit, Colorimetric Cat. No. 207007 Introduction Calcineurin (CaN) is a neuronal form of the widely distributed Ca
33VASTVNGATSANNHGEPPS51PADARPR58
Pro-rich region Trans-membrane region 214 246 359 381 UL50 1 397 211SSRTAS216PPPPPR222 NLS CR1 CR2 CR3 CR4 UL53 1 376 11RERRS15ALRS19LLRKRRR25 33VASTVNGATSANNHGEPPS51PADARPR58 FIG S1. UL97 phosphorylation
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/10/471/eaah5085/dc1 Supplementary Materials for Phosphorylation of the exocyst protein Exo84 by TBK1 promotes insulin-stimulated GLUT4 trafficking Maeran Uhm,
The topic: Molecular and Cellular Basis for Life
What is expected of you? MOLECULAR AND CELLULAR BASIS OF LIFE Professor Luke O Neill School of Biochemistry and Immunology Email: laoneill@tcd.ie Dr Suzanne Cloonan School of Biochemistry and Immunology
LANCE Eu-W1024 ITC Chelate & Europium Standard AD0013 Development grade
AD0017P-4 (en) 1 LANCE Eu-W1024 ITC Chelate & Europium Standard AD0013 Development grade INTRODUCTION Fluorescent isothiocyanato-activated (ITC-activated) Eu-W1024 chelate is optimized for labelling proteins
RayBio Human PPAR-alpha Transcription Factor Activity Assay Kit
RayBio Human PPAR-alpha Transcription Factor Activity Assay Kit Catalog #: TFEH-PPARa User Manual Jan 5, 2018 3607 Parkway Lane, Suite 200 Norcross, GA 30092 Tel: 1-888-494-8555 (Toll Free) or 770-729-2992,
DELFIA Tb-N1 DTA Chelate & Terbium Standard
AD0029P-1 (en) 1 DELFIA Tb-N1 DTA Chelate & AD0012 Terbium Standard For Research Use Only INTRODUCTION DELFIA Tb-N1 DTA Chelate is optimized for the terbium labeling of proteins and peptides for use in
Muscle Tissue and Motion
Muscle Tissue and Motion Bởi: OpenStaxCollege Muscle tissue is characterized by properties that allow movement. Muscle cells are excitable; they respond to a stimulus. They are contractile, meaning they
EFFECTS OF SIGMA RECEPTOR LIGAND BD737 IN RAT ISOLATED HEARTS
SCRIPTA MEDICA (BRNO) 80 (6): 255 262, December 2007 EFFECTS OF SIGMA RECEPTOR LIGAND BD737 IN RAT ISOLATED HEARTS Nováková M. Department of Physiology, Faculty of Medicine, Masaryk University, Brno, Czech
HDAC Fluorometric Activity Assay Kit
HDAC Fluorometric Activity Assay Kit Item. 10011563 Customer Service 800.364.9897 * Technical Support 888.526.5351 www.caymanchem.com TABLE OF CONTENTS GENERAL INFORMATION 3 Materials Supplied 4 Precautions
1. Cardiomyocytes and nonmyocyte. 2. Extracellular Matrix 3. Vessels שאלה 1. Pathobiology of Heart Failure Molecular and Cellular Mechanism
Pathobiology of Heart Failure Molecular and Cellular Mechanism Jonathan Leor Neufeld Cardiac Research Institute Tel-Aviv University Sheba Medical Center, Tel-Hashomer שאלה 1 התא הנפוץ ביותר (75%~) בלב
1- Which of the following statements is TRUE in regards to eukaryotic and prokaryotic cells?
Name: NetID: Exam 3 - Version 1 October 23, 2017 Dr. A. Pimentel Each question has a value of 4 points and there are a total of 160 points in the exam. However, the maximum score of this exam will be capped
Item Catalog Number Manufacturer 1,4-Dithioerythritol (1 g) D9680 Sigma-Aldrich
SOP: Nuclei isolation from fresh mouse tissues and DNaseI treatment Date modified: 01/12/2011 Modified by: E. Giste/ T. Canfield (UW) The following protocol describes the isolation of nuclei and subsequent
supplementary information
Figure S1 Nucleotide binding status of RagA mutants. Wild type and mutant forms of MycRagA was transfected into HEK293 cells and the transfected cells were labeled with 32 Pphosphate. MycRagA was immunoprecipitated
Minjia Tan, Ph.D Shanghai Institute of Materia Medica, Chinese Academy of Sciences
Lysine Glutarylation Is a Protein Posttranslational Modification Regulated by SIRT5 Minjia Tan, Ph.D Shanghai Institute of Materia Medica, Chinese Academy of Sciences Lysine: the most frequently modified