FDSS Applications. Optogenetic. New High Resolution Camera. New Screening Technology. Cardiovascular Disease. Antibody.
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1 Optogenetic Cardiovascular Disease New Screening Technology New High Resolution Camera Safety/Toxicology Antibody Cell Health and Energy Metabolism GPCR Enzymatic Assay High Speed Acquisition Protein-Protein Interaction Ion Channel
2 Cell Health and Energy Metabolism Oxidative Stress Assay (ROS) Assays for measuring real time production of ROS (Reactive oxygen species) as an indicator of cell heath or signalling events Energy Metabolism Assay (NADPH) NAD(P)H molecules are important cofactors for different enzymes involved in cellular pathways. Measurement of this molecule can determine metabolic activity for cells affected by disease. Research & Clinical application Inflammatory disease Monitoring inflammation of transplant before surgery Cancer (drug evaluation studies for Chemotherapy Amplex red (Thermofisher)/Luminol/ ROS-Glo (Promega) NADPH-Glo/ Pholasin -based ABEL (Knight Scientific) Publication: Application Note Nr. 25 (CBCS, Karolinska Institutet, University of Gothenburg) Identification of small molecule modulators of reactive oxygen release in TNF-α primed primarx human neutrophils. In the test system freshly isolated primary human neutrophils are first incubated with TNF-α. The cells are then triggered with cytochalasin B. The superoxide anions produced are measured by a chemiluminescence technique based on isoluminol using the single photon counting camera of the Hamamatsu FDSS 7. Addition of TNF-α/compound treated cells to pre heated Cytochalasin B (13 µl) Recording signal for 5 minutes Protein-Protein Interaction (PPI) Living-cell PPI assay is based on BRET (Bioluminescent Resonance Energy Transfer) screening technology Drug discovery BRET1 (Rluc8) (96/384), BRET2 (96 format only), NanoBret (Promega), NanoBit (Promega) 2
3 Safety/Toxicology and Cardiovascular Disease Drug discovery, Safety pharmacology (Cardiac & Neuron), Assessment of cardiotoxocity and drug efficacy early in drug development process [Ca 2+ ] sensitive dye (Cal52, ATT Bioquest)/Voltage sensitive dye (FluoVolt, Molecular probes) Any acute drug effect involved in primary or human ipsc-derived cardiomyocytes can be detected by monitoring [Ca 2+ ] transients and electrical activity. Application Note Pluricyte Cardiomyocytes.1 % DMSO 1 µm Diltiazem 1 µm Bay K 8644 : 1: 1 nm E 431 : 1: 1 µm Nifedipine : 1: 1 nm Isoproterenol : 1: : 1: : 1: Application Note COR.4U Application Protocol icell Cardiomyocytes2 A B C Baseline Isoproterenol (1 µm) E-431 (1 µm) 1, RFU 5, Time (sec) Time (sec) Time (sec) Presentation 3 rd FDSS application Workshop, Barcelona, Stephane Bedut (ICM, Servier) Membrane potential activity hips-derived cardiomyocytes in 96 wells Ivabradine 1 µm Lidocaine 5 µm 1 1 F.u. 1 F.u. 1 s 1 s Acquired 1 s (black) and 3 min. (red) after ouabain treatmenz (96 format, FluoVolt dye) Diastolic slope (F.u.s -1 ) Vehicle Ivabradine 1 µm Ivabradine 2 µm Lidocaine 5 µm 3
4 GPCR GPCRs (G-protein-coupled receptors) are one of the most important classes of drug targets Effect of GPCR pathway activation: intracellular Ca 2+, camp, Beta Arrestin Drug discovery [Ca 2+ ] sensitive dye (Cal52,ATT Bioquest)/Voltage sensitive dye (FluoVolt, Molecular probes), BRET, camp (Glow sensor,promega), Beta Arrestin (Discoverx) Recording of Calcium transients using the Hamamatsu μcell: Response to neurotransmitter (a) Representative traces from calcium transients induced by various neurotransmitters, KCl as positive control and thyrode solution as negative control are shown. The traces are normalized to the baseline fluorescence. (b) The peak of calcium transients are calculated and plotted agaínst the neurotransmitter concentration. (a) 1.4 Normalized to baseline fluorescence Thyrode solution KCl Glutamate NMDA Acetylcholine Histamine Dopamine Serotonin ATP Time [s] (b) 1.4 Normalized to baseline fluorescence GLutamate NMDA Acetylcholine Dopamine Serotonin Histamine ATP Dopamine 1µM Aripiprazole 1µM Thyrode solution (negative Bifeprunox control) 1µM KCl (positive control) Quinpirole 1µM Concentration [μm] Publication: FDSS Application Nr. 23 (a) Antagonism Dopamine 1 µm (b) Dose-response Dopamine (C) Efficacies Haloperidol 1 µm EC5=7.3 + Dopamine 1 µm Haloperidol 1 alone Log [ligand] (M) -5 BRET (% vs Dopamine 1 µm) BRET (% vs Dopamine 1 µm) BRET induced by compounds (% vs Dopamine 1 µm) Dopamine 1µM Aripiprazole 1µM Bifeprunox 1µM Quinpirole 1µM The antagonist Haloperidol fully reverses the BRET signal induced by dopamine (a). The stimulation by dopamine is dose-dependent (b). Dopamine efficacy can be discriminated from other compounds activity (c). Print screen FDSS software GPCR assay: 1536 assay Ca 2+ Aequorin B2-Arrestin 2 Recruitment n Full agonist vs. partial agonist: w/o full view > misleading n Efficacy vs. AUC vs. Slow binders > kinetics n Time course is key Cpd1 Cpd2 Cpd3 4
5 Ion Channel Publication: YFP-halide assays for CFTR drug discovery using the FDSS/μcell Thierry Christophe, PhD, Director - Biology, Galapagos NV Hamamatsu 11 th FDSS User Meeting 11 June 215 Detection of CFTR potentiators Cells over-expressing F58d-CFTR and YFP-H148/I152L : Drug discovery Fsk + CFTR potentiator Cl- Cl- Cll- l- l- l- Na+/K+ channel: TEFLAB dye, Membrane potential dye (Molecular device)/fluxor Potassium Ion Channel Assay (Thermo Fisher)/SBFI (Thermo Fisher)/ FRET VSP dye (Invitrogen) Cl- channel Fluorescence of Yellow Fluorescent Protein mutant (YFP-H148Q/I521L) quenched by halides F/F NaI time Vehicle/inactive CFTR potentiator Fsk + inactive cpd 1-F/F Dose response CFTR potentiator log conc (M) Antibody (Functional Assay) Drug discovery (ion channel/gpcr) Ca 2+ dye 8 nm Agonist Antibody Ca 2+ buffer only Calcium Mobilization response: Inhibition of Ca 2+ response induced by EC8 of Agonist in presence of different antibodies Enzymatic Assay (Isomerase Inhibitor Screening) Use of a Real-Time Fluorescence Monitoring System for High- Throughput Screening for Prolyl Isomerase Inhibitors Tadashi Mori, Selma Itami, Tomotaka Yanagi, Yota Tatara, Mari Takamiya and Takafumi Uchida Conversion Ratio (%) CypA none, CsA none CypA 7 nm,csa none CypA 7 nm, CsA 3.2 nm CypA 7 nm, CsA 12.5 nm CypA 7 nm, CsA 5 nm CypA 7 nm, CsA 2 nm The K m value of CypA with the peptide was 59 M. (C) Concentration dependence of CsA inhibition of CypA activity. Concentrations of CypA and CsA are expressed as follows: CypA none, CsA none ( ); CypA 7 nm, CsA none ( ); CypA 7 nm, CsA 3.13 nm (x); CypA 7 nm, CsA 12.5 nm ( ); CypA 7 nm, CsA 5 nm, ( );and CypA 7 nm, CsA2 nm ( ). 5
6 New Screening Technology Electric field Stimulation (EFS) 96-channel electrode array Stimulate all 96 wells simultaneously Cylindrical electrodes Stimulation voltage is changeable column by column Cardiac, CNS, Muscular diseases Ca 2+ dye/(fdss/μcell + EFS + High speed options) Caution Notice: The FDSS/µCELL EFS system should not be used for optically detecting/monitoring change in transmembrane potential of the cells. The FDSS/µCELL EFS system should not be used on any cell or cells in which the user or anyone else has expressed target ion channels. High-Throughput Fluorescence Measurements of Ca 2+ Transients in primary rat and in human ipsc-derived cardiomyocytes (a) Rat primary cardiomyocytes P rate [/min] P rate [/min] N=96 without 1. 1.H Hzz a stimulation stimulation Spontaneous beating With 1. Hz stimulation (b) Ca 2+ channel blocker: Verapamil a) Primary rat cardiomyocytes (Cosmo Bio) were cultured in 96-well plate. The above figures show the intracellular Ca 2+ concentration changes for 5 s in 96 wells in a microplate. In primary cultured cardiomyocytes, cells in each well beat at different rates and with different timing (left). When electrical field stimulation was applied (1. Hz, voltage 5 V, duration 5 ms), the result was uniform Ca 2+ oscillations between all wells resulting in synchronized beating of cells (right). b) Effects of ion channel blockers on Ca 2+ transients in ips cardiomocytes: Beating rate is dependent on Electric Field Stimulation frequency. Electric Field Stimulation (EFS) of human ipsc-derived neuron using Hamamatsu FDSS/μCELL icell Neurons The Ca 2+ response is inhibited in the presence of a calcium channel blocker, Bepridil.1 Fluorescence intensity ( F/F ).5 Amplitude ( F/F ) IC5=5.6 µm Time (s) Bepridil [M] Bepridil, a calcium channel blocker, was added to the wells at the indicated concentration and incubated for 2 min, and then EFS pulses (5 ms of pulse width, 4 Hz) were given. The figures on the right show intracellular Ca 2+ concentration changes. At more than 15 μm, the Ca 2+ response was completely inhibited. The IC 5 value was estimated to be 5.6 μm (graph on the left). Presentation CDI 12 th FDSS Users Meeting 2V 3V 4V 5V 6V icell Skeletal Myoblasts icell SkM + EarlyTox calcium dye Compound Treatment Electric Field Stimulation (EFS) 6
7 Optogenetic Rhodopsins: Light-gated ion channels Rhodopsins: Light-gated ion channels ChR2 (D156A) : Drug discovery Detection system: Ca 2+ sensitivite Fluo8 No Wash dye Stimulus: Depolarization induced by ChR2 activation with blue light pulses Target: human Cav1.3 (α1/α2δ1/β3) Recipient cell line: HEK-293/kKir2.3 Detection system: Ca 2+ sensitive Fluo8 No Wash dye Stimulus: depolarization induced by ChR2 activation with blue light pulses Cav1.3 Blocker: Isradipine HTS instrumentation: Hamamatsu FDSS/µcell Blue light ChR2 induced membrane depolarization (τ-off 6.9 min) Other information: FDSS/μCell & FDSS7 High Speed Acquisition & Fast Camera Option High speed acquisition Application: Enzymatic, Optogenetic, Cardiac assay... Speed of acquisition: 2 Hz (96 well) 1 Hz (384 well) New High Resolution Camera ( Well Analysis ) Measurement 3D Cardiomyocyte (Spheroid) Kuraray spheroid microplate (cardiomyocyte) 1 pixel around 1 spheroid 1 pixel data showed Ca 2+ oscillation of each spheroid. Analyzed using Hamamatsu Software, AQUACOSMOS 1 s 7
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