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Multi-Protein Detection of Endothelial Cell Inflammation and Single Cells' Heterogeneity Study

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Multi-Protein Detection of Endothelial Cell Inflammation and Single Cells' Heterogeneity Study University of New Hampshire University of New Hampshire Scholars' Repository Master's Theses and Capstones Student Scholarship Fall 2012 Multi-protein detection of endothelial cell inflammation and single cells' heterogeneity study Jingwen Chai University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/thesis Recommended Citation Chai, Jingwen, "Multi-protein detection of endothelial cell inflammation and single cells' heterogeneity study" (2012). Master's Theses and Capstones. 728. https://scholars.unh.edu/thesis/728 This Thesis is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Master's Theses and Capstones by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. MULTI-PROTEIN DETECTION OF ENDOTHELIAL CELL INFLAMMATION AND SINGLE CELLS' HETEROGENEITY STUDY by Jingwen CHAI B.S., Tianjin University of Science and Technology, 2010 THESIS Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Master of Science in Chemical Engineering September, 2012 UMI Number: 1521547 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI 1521547 Published by ProQuest LLC 2012. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. uest ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 This thesis has been examined and approved. 7^ Thesis Director,> Dr. QinjQing Song Assistant Professor of Chemical Engineering Dr. Russell Carr Professor of Chemical Engineering Dr. ivfivedita Gupta Associate Professor of Chemical Engineering Date ACKNOWLEDGEMENTS I would like to thank Dr. Song for her thorough guidance, encouragement and support throughout my graduate study. Thanks to Dr. Carr and Dr. Gupta for kindly serving on my committee. I would also like to thank Dr. Mark Townley for training me with the usage of Zeiss confocal microscope and giving us many helpful suggestions during the research. Thank Dr. Andrew P. Laudano for letting us use his lab. Finally, I would like to thank my family and friends for their support and love. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS iii LIST OF TABLES . viii LIST OF FIGURES ix ABSTRACT xii Chapter 1 INTRODUCTION 1 1.1 Background of the study 1 1.2 Multiple-protein detection 2 1.2.1 Proteins picked in the study 2 1.2.2 The technique used to detect multiple proteins 4 Chapter 2 QUANTITATIVE AND MULTIPLEXED STUDY OF ENDOTHELIAL CELL INFLAMMATION 6 2.1 Introduction 6 2.2 Experiment materials and methods.... 9 2.2.1 Chemicals 9 2.2.2 Human aortic endothelial cell culture 10 2.2.3 Multiplexed immunofluorescence staining 10 2.2.4 Mean fluorescent intensity (MFI) measurements 11 2.3 Results 11 2.3.1 Autofluorescence and specificity of mAb binding 11 2.3.2 Optimum concentrations of mAbs 12 iv 2.3.3 Quantitative and multiplexed protein detection to characterize dynamic responses by HAECs to inflammatory stimulation 15 2.4 Discussion 22 Chapter 3 MULTI-SPECTRAL CONCURRENT DETECTION OF MULTIPLE PROTEINS 25 3.1 Introduction 25 3.2 Experiment material and methods 30 3.2.1 Chemicals 30 3.2.2 Human aortic endothelial cell culture 30 3.2.3 Customer-conjugation of purified antibodies 31 3.2.4 Multiplexed immunofluorescence staining 31 3.2.5 Preparing poly-lysine glass slides 32 3.2.6 Reference spectrum 32 3.2.7 Cross-reactivity of monoclonal antibody (mAb) 32 3.3 Results 33 3.3.1 Single protein detection by conventional band-filter technology....33 3.3.2 Reference spectrum 34 3.3.3 Multispectral imaging of multiple proteins and linear unmixing....35 3.4 Discussion 46 Chapter 4 MULTIPLE-PROTEIN DETECTIONS OF SINGLE-CELLS REVEAL CELL-CELL HETEROGENEITY IN HUMAN CELLS 49 4.1 Introduction 49 v 4.2 Experiment material and methods 52 4.2.1 Materials and Chemicals 52 4.2.2 Human aortic endothelial cell culture 53 4.2.3 Multiplexed immunofluorescence staining 53 4.2.4 Mean fluorescent intensity (MFI) measurements 54 4.2.5 Histogram of protein expression in a cell population 54 4.2.6 Noise (cjjand a,) 55 4.2.7 Noise strength (9; and <p0 55 4.2.8 Correlation coefficient R2 55 4.3 Results 56 4.3.1 Multiple protein detection 56 4.3.2 Histogram of protein expression in a cell population 58 4.3.3 Noise and noise strength 61 4.3.4 Correlation coefficients R-J 66 4.4 Discussion 71 Chapters CONCLUSIONS AND FUTURE WORK 74 5.1 Conclusions 74 5.2 Future work 75 REFERENCES 77 APPENDIX A: MEAN FLUORESCENT INTENSITY DATA FOR CHAPRTER 2 83 vi APPENDIX B: MEAN FLUORESCENT INTENSITY DATA FOR CHAPRTER 4 vii LIST OF TABLES 4.1 - Correlation coefficients of TLR2, TLR4 and CD62P expressed on same single cells 69 4.2 - Correlation coefficients of CD54, CD105 and CD62E expressed on same single cells 71 A.l - Effects of inflammatory stimulation on proteins expression 83 B.l - MFI data for single cells about CD62E (E-selectin) under 4 hr TNF-a stimulation 84 B.2 - MFI data for single cells about all 6 proteins under Ohr (control), 4hr, 12hr and 24hr TNF-a stimulation 98 viii LIST OF FIGURES 1.1 - Different subsets of adhesion molecules have been shown to be responsible for leukocyte rolling and arrest on the vascular endothelium by modulating CAM expression on the surface of both the endothelial cells and the circulating leukocytes [3,4] 4 2.1 - (A) Autofluorescence (red) and DAPI (nuclei, blue) image of human aortic endothelial cells (HAECs). (B) DIC image of HAECs 12 2.2 - Optimum concentrations of fluorophore-conjugated antibodies (CD62P-AF647).. 13 2.3 - Optimum concentrations of fluorophore-conjugated antibodies 14 2.4 - Multiple protein detection in HAECs. VCAM-1, ICAM-1, and E-selectin, as visualized by immunofluorescence staining, DAPI-stained nuclei, and DIC images from a quiescent HAEC preparation (A) and from a TNF-a stimulated HAEC preparation (B) 16 2.5 A - Effects of inflammatory stimulation on expression of TLR2 19 2.5B - Effects of inflammatory stimulation on protein expression of P-selectin 19 2.5C - Effects of inflammatory stimulation on protein expression of TLR4 20 2.5D - Effects of inflammatory stimulation on protein expression of E-selectin 20 2.5E - Effects of inflammatory stimulation on protein expression of ICAM-1 21 2.5F - Effects of inflammatory stimulation on protein expression of VCAM-1 21 3.1 - The principle of emission fingerprinting 29 3.2 - Single protein detection by conventional band-filter technology 33 ix 3.3 - Reference spectrums for all of the fluorescent entities 34 3.4 - Multispectral images collected as a set of two-dimensional images as a function of wavelength 35 3.5 - Concurrent three protein detection using emission fingerprinting technique 36 3.6 - Concurrent four protein detection using emission fingerprinting technique 37 3.7A - Concurrent eight protein detection using emission fingerprinting technique. Laser 488nm irradiates TLR2-AF488 (Dartmouth green) and TLR4-PE (Red) proteins.. 39 3.7B - Concurrent eight protein detection using emission fingerprinting technique. Laser 543nm irradiates RAGE-AF555 (Fandango), ICAM3-AF546 (Purple), and PECAM1-AF568 (Cyan) proteins 39 3.7C - Concurrent eight protein detection using emission fingerprinting technique. Laser 633nm irradiates E-selectin-AF700 (Taupe), P-selectin-AF647 (Orange), and ICAM1-AF660 (Brown) proteins 40 3.8A - Concurrent ten protein detection using emission fingerprinting technique. Laser 488nm irradiates TLR2-AF488 (Dartmouth green) and TLR4-PE (Red) proteins.. 41 3.8B - Concurrent ten protein detection using emission fingerprinting technique. Laser 543nm irradiates RAGE-AF555 (Fandango), ICAM3-AF546 (Purple), PECAM1- AF568 (Cyan), and VCAM1-AF594 (Olive) proteins 42 3.8C - Concurrent ten protein detection using emission fingerprinting technique. Laser 633nm irradiates NF-KB-AF680 (Violet), and E-selectin-AF700 (Taupe), P-selectin- AF647 (Orange), and ICAM1-AF660 (Brown) proteins 42 3.9A - Representative higher magnification images in Figure 3.8A 43 x 3.9B - Representative higher magnification images in Figure 3.8B 43 3.9C - Representative higher magnification images in Figure 3.8C 44 3.10A - Representative higher magnification images in Figure 3.8A 44 3.10B - Representative higher magnification images in Figure 3.8B 45 3.IOC - Representative higher magnification images in Figure 3.8C 45 4.1 - Multiple protein detection on HAEC cells using fluorescent microscope 57 4.2 - Cell-cell variability in CD62E expression levels in a cell population 57 4.3 - CD62E (E-selectin) protein distributions (histogram) as a function of cell numbers 58 4.4 - Dynamic behaviors of histograms of TLR2, TLR4, CD62P, CD54, CD106, and CD62E 60 4.5 - Noise, noise strength of six protein histogram as a function of stimulation time.... 62 4.6 - Noise, noise strength of independent cell sub-sets at 4hr of TNF-a stimulation 65 4.7 - correlation coefficients of multiple proteins (TLR2, TLR4, and CD62P) on the same single cells 68 4.8 - correlation coefficients of multiple proteins (CD54, CD105 and CD62E) on the same single cells 70 xi ABSTRACT MULTI-PROTEIN DETECTION OF ENDOTHELIAL CELL INFLAMMATION AND SINGLE CELLS' HETEROGENEITY STUDY by Jingwen CHAI University of New Hampshire, September 2012 Endothelial inflammation plays major roles in all phases of atherosclerotic process and both innate immunity and endothelial adhesion molecules contribute to endothelial inflammation.
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