Anmis ImageStream Mk II imaging flow cytometer for ONR funded research
Abstract
We are requesting funds for the purchase of a Luminex Corporation Anmis ImageStream Mk II fluorescence imaging flow cytometer with t,he FlowSight Systems high gain capability to expand our ability to discern the pathophysiology of extracellular vesicles (EVs) in ON,R funded research. EVs are found in all body fluids and increase in association with virtually every human disease and injury. Two t,ypes play roles in cell-to-cell communication in normal health and in pathological conditions, exosomes and microparticles (MPs). Th,e technology for studying these components has evolved over the past decade and with it, improved understanding of their roles in he,alth and disease. A major impediment to this work is the size of the particles. Exosomes are ~20-120 nm diameter particles generated, by the endosomal pathway and MPs are 100-1000 nm particles generated by an outward budding of plasma membrane. Over the past 10 yea,rs we have elucidating roles for blood-borne EVs in decompression sickness,stresses related to breathing elevated levels of carbon d,ioxide, high and low pressure oxygen, and in several other disorders using flow cytometry for MPs analysis and tunable resistance pu,lse sensing (TRPS) or nanoparticle tracking analysis (NTA) for studying exosomes. There is no firmly established approach for EVs a,nalysis. The International Society for Extracellular Vesicles has acknowledged this and stated that no current isolation protocol ca,n purify based on biogenic origin or size alone. They recommend that EVs are described based on physical characteristics, biochemica,l characteristics and cell of origin or stimulus condition. We have carried out these components in our research and published 32 pe,er reviewed papers on detection methods, production mechanisms and pathophysiol,re well described EV isolation methods, each approach carries risks of missing some EV types. Therefore, detection methods need to b,e sensitive but also avoid steps that may exclude some EV subtypes. The Luminex instrument has numerous advantages over our current,apparatus. Moreover, as we have 29,000+ hours operation on our instrument, it is approaching the end of useful life despite continuo,us maintenance under the manufacturer service agreement. EVs are characterized based on physical characteristics, omics (transcip,tomic, genomic, proteomic ad lipidomic analysis) and functional assays. Isolation by ultracentrifugation and imaging by electron mic,roscopy are, in some respects, gold standards but the artifacts these methods impose have raised concerns that results may not be,representative of EVs in the real world. We will offer representative examples below for these and other methods from our own rese,arch. A core issue, and the reason we are seeking funds for the Luminex instrument, is that an experimental approach that requires m,inimal manipulation of samples, combines physical with phenotypic analysis of all sizes of EVs, and has capacity to sample a large n,umber of particles has great advantages. The ImageStreamX Mk II with high gain option combines all these characteristics in a single, platform. Numerous flow cytometers and several types of NTA devices are available on campus, but nothing with the capabilities of t,he Luminex instrument. We have shared our plans with other University investigators (see attached letters) because instrument availa,bility to graduate students across the campus will provide access to the very latest in cutting-edge technical capabilities. This in,strument will extend our ability to educate future scientists through research important to DoD missions.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Apr 01, 2022
- Source ID
- N000142212369
Entities
People
- Stephen R. Thom
Organizations
- Office of Naval Research
- United States Navy
- University of Maryland