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  1. 1. A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents X. Zhu, A. Fu, K. Q. Luo, 2012 ILANA J PORTER An early high- throughput application of GFP-FRET
  2. 2. The importance of high-throughput • Automation • Can conduct thousands of assays • Drug discovery http://www.penchovsky.site90.com/re search.php
  3. 3. FRET - Fluorescence Resonance Energy Transfer Donor fluorophore is excited by light pulse Energy is transferred to acceptor fluorophore Acceptor fluorophore emits light Citation for image
  4. 4. Importance of FRET measurements • High spatial resolution (10 nm) • Non-disruptive to cells • Real-time measurements of living cells Previous applications •Protein conformations •Identifying signaling pathways •Protein-protein interactions
  5. 5. and Caspace-3 • Increased apoptosis associated with atherosclerosis • Inhibition of apoptosis stimulates capillary growth • Solid tumor growth and metastasis • Possible target for tumor therapy • Primary execution protease • Cleavage sequence Asp-Glu-Val-Asp (DEVD) Endothelial Apoptosis http://commons.wiki media.org/wiki/File: Caspase_3.png
  6. 6. C3 FRET Sensor • Analog for Caspace-3 substrate • Fusion of FRET donor and acceptor • Cleavage disrupts FRET CFP Donor D E V D YFP Acceptor
  7. 7. Initial FRET Measurements Citation here
  8. 8. Moving to High-Throughput • 96-well plate • Minimum FRET levels after 12 hours UV • FRET does not reduce from necrotic inducers But only detects cleavage of substrate analog, not actual substrate Are these measurements accurate? Could there be competition?
  9. 9. Checking the Method: MTT Assay Citation here
  10. 10. Checking the Method: Activity Assay Citation here
  11. 11. Checking the Method: Western Blot Citation here
  12. 12. Application to Paclitaxel Citation here CC50 = 1.2 nM FRET50 = 0.71 nM
  13. 13. Paclitaxel Application 2 Citation here
  14. 14. High-throughput Reliability • Z’ factor evaluates quality of high-throughput assay • Z’>0.5 is standard • Z’ = 0.66 for the FRET assay Citation here
  15. 15. First Test of the Assay: Dioscin Citation here
  16. 16. Issue with Comparison • CC50 is standard measurement for cytotoxicity • FRET50 is more sensitive How can results from this assay be compared to previous research?
  17. 17. FRET is perfect for High-throughput • Distinguish apoptosis • Non-disruptive to live cells • Z’ indicates reliable high-throughput method • High sensitivity
  18. 18. Future Applications of this Screen • Co-culture • Endothelial protective drugs • Prevent vascular Injury • Screening of different cell lines
  19. 19. Thank You Questions?

Editor's Notes

  • -Widely used tool of biochemists is high-throughput assay-utilizes robotics and automated plate-readers to perform thousands of experiments, automated.-Generally used for discovery of drugs
  • -FRET stands for-consists of two fluorophores, light emitting and absorping protein moieties-GFP derivatives-Distance dependent, see image-FRET efficiency is acceptor emission over donor emission, y/c
  • -FRET is an extremely powerful spectroscopic tool-Energy transfer only occurs when fluorophores are less than 10 nm apart-Extremely high spatial resolution, good temporal resolution-GFP and derivatives non-disruptive to protein conformations when inserted correctly, non toxic-Can be applied to in vivo measurements
  • -Endothelial cells line interior surface of blood vessels-Too much apoptosis occurs, atherosclerosis and thrombosis-Apoptosis is inhibited, stimulate growth of capillaries, which allows tumors to grow & metastasize-Endothelial cells are a target for tumor therapy-caspase 3 is an important protease involved in apoptosis
  • -C3 is a recombinant substrate for caspace 3-two fluorophores, cyan donor and yellow acceptor-connected by cleavage sequence-cleave c3, no FRET, signal changes from green to blue-Transformed into human umbilical vein endothelial cells
  • -irradiated cells for 3 min under UV light-incubated for different periods, then took FRET measurements-cells turn from green to blue, c3 is cleaved-shape becomes round and shrink, apoptosis-compare to paclitaxel, anticancer drug
  • -performed with 96-well plate, at same time and automated-shine UV for 3 minutes, decrease FRET after 3 hours-minimum FRET after 12 hours-necrotic inducers H2O2, cell death but not apoptosis-necrotic does not reduce FRET, no C3 cleavage -but detect analog --- oops
  • -MTT assay biochemistry method-determines cell viability, percent-longer UV incubation, lower FRET and lower Viability-for necrotic factors, FRET does not change but viability decreases-this FRET assay can specifically detect apoptosis not necrosis
  • -performed an activity assay of caspace 3-to prove that it was activated during UV apoptosis-not activated H2O2 necrosis
  • -western blot visualize proteins in cell-specific proteins stick to antibodies-PARP endogenous substrate for caspace 3-c3 is cleaved at the same time
  • -Pax is a known cancer drug-fig A dose dependent MTT cell viability assay-cc50 is cell cytotoxicity, 50% of cells die-fig B is dose dependent FRET emission ratio-FRET50 , 50% reduction, is lower. FRET is more sensisitve
  • -time dependent FRET emission for 3nM dose and 30nM dose-30nM has faster reduction of FRET-able to compare potency
  • -Z factor measures quality of high-throughput assay-this assay is reliable -Z = 1- [(3sigs + 3sigc)/|mus-muc|] stimulated and control
  • -applied the assay to various compounds they had-found dioscin, herbal compound-fig A dioscin reduced FRET in a dose and time dependent manner-fig B caspace 3 activity assay, activity enhanced by dioscin-fig C western blot, PARP cleavage is induced
  • -cc50 standard measurement-FRET50, new statistic introduced here-hard to compare to previous research-Can’t estimate dosage
  • -This assay can distinguish apoptosis from necrosis-can be performed in live cell-z’ indicated reliability of the method-fret measurement FRET50 more sensitive than previous methods
  • -co-culture means multiple types of cells-can distinguish these signals and measure them separately-measure opposite, anti-apoptotic drugs-prevent vascular injury, two possible causes-use a different cell line other than human umbilical vein endothelial cells
  • -co-culture means multiple types of cells-can distinguish these signals and measure them separately-measure opposite, anti-apoptotic drugs-

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