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Flow Cytometry at the University of New Mexico

by Larry Sklar

Flow Cytometry at the University of New Mexico has its roots in the development of the National Flow Cytometry Resource (NFCR) at Los Alamos National Laboratories (LANL). Noel Warner, a professor in the College of Pharmacy at UNM was an early adopter of flow cytometric approaches in immunology and traveled regularly from Albuquerque to Los Alamos in the 1970’s and 1980’s to collaborate with the team of physicists and engineers who were developing flow cytometric platforms. To this day, there are stories about how Noel and his team of postdocs and grad students hurried back and forth over the 100 mile journey without getting speeding tickets. Flow cytometry became a mainstay of the immunology community at UNM.

The UNM Shared Flow Cytometry Resource has been in existence for more than 20 years. In a sense, the resource was born with the award of an NIH shared instrument grant for a Coulter 753 cell sorter to Janet Oliver in 1988. The UNM Cancer Research and Treatment Center recruited Larry Seamer, then at Stanford, to manage the core facility. Larry Sklar was jointly recruited from Scripps by UNM and LANL in 1990 to build bridges between UNM’s School of Medicine and the NFCR. Given his training in physical chemistry, cell biology, and immunology, Sklar was particularly interested in developing rapid mix or stopped flow (subsecond) flow cytometry capabilities for exploring signal transduction, and taking advantage of the ideas at Los Alamos for spectroscopic analysis of molecular interactions in flow cytometric systems. Seamer spent part of his time at UNM exploring automated sample handling systems at UNM before moving to Bio-Rad.

The flow cytometry core at UNM was originally located in the basement of the main Cancer Research and Treatment Center (CRTC) Building. This building had been constructed in the 1970’s to house an NCI designated Cancer Center that would partner with Los Alamos in the investigation of pi-mesons for cancer therapy.

UNM and LANL Collaborators at Angel Fire, Rocky Mountain Cytometry

When Larry Seamer joined BioRad in 1995, the facility recruited Bruce Edwards as a faculty member to manage the core facility and to serve as a partner in the development of new sampling handling systems. Sklar and Edwards realized that repetitive rapid measurements of cell responses and molecular interactions on cells and beads could provide a role for flow cytometry in high throughput screening and drug discovery. Under the direction of Edwards, the shared resource moved to the second floor of the new Cancer Research Facility (CRF) Building when it opened in 1999.

The UNM shared flow cytometry resource has had a significant innovation component since 1990 when Sklar came to New Mexico to direct both the shared resource and the NFCR. As Sklar arrived in New Mexico with his own research flow cytometers, there has been a continuous interplay of technology development and biological application among CRTC research programs, LANL NFCR, and the shared facility. Over this period, there have been more than 40 joint publications with LANL. This interplay was further enhanced by the arrival of Edwards, whose technological, biological and administrative skills complemented Sklar’s. Through a series of internal collaborations, industrial partnerships and NIH grants, we developed both subsecond time resolution and high throughput flow cytometers. We have applied the instruments to cells and particle-based molecular assemblies. These developments involved both the fee-for-service cytometers of the shared resource and the research cytometers that Sklar and Edwards maintained through their own research grants.

In 1999, in collaboration with Axiom Biosciences, Sklar and Edwards developed hardware and software for our first generation of high throughput flow cytometry, Plug Flow Cytometry (Cytometry 37:156-159, 1999). This technology development contributed research applications for NIH funding for separate cell signaling (NIH Bioengineeriing Research Partnership BRP GM60799/EBO00264) and cell adhesion grants (RR14175/EB02022) in 1999. Interestingly, both grants moved from their original institutes to NIBIB as founding grants. The idea that this technology has biological applications was validated when the resubmission of the adhesion grant was reassigned by NIH as HL081062 with funding anticipated this July, 2007. This work on cell adhesion (Alexandre Chigaev, Tione Buranda, Gordon Zwartz and Richard Larson) has developed sample delivery systems which shear cells as well as fluorescent ligands that bind to integrins. These ligands have been used in flow cytometry to understand the relationships among ligand affinity, cell adhesion and integrin conformation. The conformational analysis developed real-time fluorescence resonance energy transfer methods.

A patent was awarded in 2001 to Sklar, Edwards and Fritz Kuckuck for the Plug Flow Cytometry technology. During this same year the development of a second generation of high throughput flow cytometry technology was reported and subsequently designated as HyperCyte (Cytometry 44:83-90, 2001). The patent was approved in 2004. The successful prototype of a second generation high throughput flow cytometer led to a second grant BRP with Novasite Pharmaceutricals (AI48517) in 2001. This BRP, in combination with institutional funds and a partnership with DakoCytomation, allowed the group to add a MoFlo high-speed sorting flow cytometer.

Susan Young was hired for the Novasite project. This automated flow cytometry sample handling project led to the concept of a Microtechnology Laboratory in 2002 to support sample handling for multiwell plate based analyses. The microtechnology concept was approved for support through a peer-reviewed internal award process (High Throughput Flow Cytometry Screening Resource Initiative). The Microtechnology Lab acquired a Titertek MAP-C2 microplate pipetting instrument, and space for it was reclaimed from a storage room in the CRF basement. This development work and early screening on cell-based projects ultimately contributed to: an NIH Roadmap proposal for the Molecular Libraries Initiative (U54 MH074425), partnerships with Luminex and LANL NFCR for high throughput multiplexing, and a partnerships with the NFCR at LANL in the current P41 renewal (RR01315) that would be directed to developing technology for the benefit of investigators involved in Molecular Libraries Projects.

Peter Simons has played a significant role in the team for the development of bead-based assays of molecular assembly. These assays have included multiplexes of soluble transmembrane G protein coupled receptors in complexes with ligands and G proteins. More recently, we have developed multiplex assays for kinases, intact proteasome, low molecular G proteins, and Bcl-2 family members. Eric Prossnitz has played a significant role in cell and receptor molecular biology, physiology, and biochemistry.

The NIH named UNM as a Molecular Library Screening Center in July 2005 (http://nmmlsc.health.unm.edu/overview.shtml). The overall goal of the molecular libraries initiative is the development of small molecules to be used as probes, imaging agents, and potentially as leads for novel therapeutic agents. The Molecular Libraries Initiative is part of the overall NIH Roadmap vision to accelerate biomedical research and to generate new tools for discovery. The New Mexico team has been subsequently focused on the development of collaborative target projects featuring cell and bead-based assays, particularly multiplex targets. The sample handling technology has been implemented on virtually all flow cytometry platforms, and we are exploring platform improvements for performance and throughput in collaboration LANL, the Center for Biomedical Engineering at UNM, Sage Sciences and Luminex.

The Roadmap team from left to right: Cristian Bologa, Tudor Oprea, Eric Prossnitz, Susan Young, Jeff Arterburn, Bruce Edwards, Larry Sklar.

The current flow cytometry team at UNM consists of approximately 20 members involved in screening and discovery as well as basic research. Other members of the screening team include Virginia Salas, Mark Carter, Juan Strouse, Mark Haynes, Irena Ivnitski-Steele, Anna Waller, Zurab Surviladze, Danuta Wlodek, and Terry Foutz. The progress of screening of the following assays can be tracked at PubChem (http://pubchem.ncbi.nlm.nih.gov/). Cheminformatics for the screening team is lead by Tudor Oprea and Cristian Bologa. Chemical Synthesis for the screening team is led by Jeffrey Arterburn at NMSU and Alex Kiselyov at ChemDiv.

High Throughput Flow Cytometry for the NIH Roadmap
Assay NIH Status and Tracking
Cytotoxicity / 384 well plate miniaturized  
FPR / FPRL1 duplex 1 R03 MH076381-01
Allosteric Ligands for the VLA-4 Integrin affinity activation and inhibition 1 X01 MH077638-01
Ligands of GRP30 and Classical Estrogen Receptors 1 X01 MH077627-01
ATP Hydrolysis-dependent disassembly of the 26S proteasome 1 X01 MH077613-01
Androgen Mediated LnCAP Differentiation 1 X01 MH078937-01
Quorom Sensing for Bacterial Virulence 1 X01 MH078952-01
Arrestin binding to GPCR tail 1 X01 MH077637-01
Bcl-2 Family Protein Multiplex 1 X01 MH079850-01
GTPase Multiplex 1 R03 MH081231-01
Multiplexed flow Cytometry Screens for RGS inhibitors 1 R21 NS057014-01
Efflux Pump Inhibition for Leukemia Therapy In Development