FLOW CYTOMETRY, SAMPLE HANDLING AND CELL ADHESION

Summary

Principal Investigator: Larry Sklar
Affiliation: University of New Mexico
Country: USA
Abstract: Adhesion among blood cells and vascular endothelial cells (EC) is important in health and disease: for example, neutrophils participate in host defense, acute inflammation and reperfusion injury, monocytes and lymphocytes in chronic inflammation and immunological diseases, eosinophils and basophils in allergy and asthma, and platelets in coagulation and thrombosis. The interactions among circulating cells and EC are a sequence of disease specific traffic signals. The signals target the circulating cells to particular vascular sites by allowing a weak rolling attachment and activate the cells to allow firm attachment and migration into tissues to participate in the disease process. In metastasis, tumor cells recapitulate the steps of the leukocytes. At the molecular level, firm attachments are typically protein-protein interactions mediated by integrins and ligands called ICAMs and VCAMs. Weaker interactions are mediated by protein-carbohydrate binding between selectins and carbohydrate-bearing cellular mucins. Relatively little is known about the intrinsic affinities, association and dissociation rate constants, or molecular changes which allow the interactions to occur as needed but not among resting cells. At the cellular level, the molecular affinity translates into the avidity of adhesion. On surfaces, weaker interactions allow cells to roll under the shear forces of blood whereas the firm attachments nearly immobilize adherent cells. Specific receptors allow cells in suspension or surfaces to adhere following a collision. All in all, vascular targeting of blood cells under shear depends upon integrating cellular activation and adhesion molecule conformation with high and low affinity molecular recognition steps. Some approaches to examine molecular recognition, cell adhesion and cell activation are available. Molecular interactions between purified molecules can be examined with surface plasmon resonance with a time resolution of a few seconds. Cellular adhesive interactions under shear in vivo and in vitro can be observed by microscopy. Flow cytometry is already an important tool for studying cell activation. Recently, the flow cytometry repertoire has added analysis Of molecular interactions with sub-second resolution, cellular adhesion under shear, and cell detection in whole blood. We propose to apply and extend our recent advances in on-line sample handling for flow cytometry, with off the shelf components, to cell adhesion. We will, for the first time, apply flow cytometry with sub-second resolution to cell adhesion. We will optimize cytometry for binding between cell and soluble adhesion molecules. We will integrate automated cytometry with cone-plate viscometry to achieve on-line analysis of cellular interactions under shear. We will integrate flow cytometry with viscometry to analyze molecular interactions under shear. The multi-parameter capabilities of flow cytometry will uniquely enable the simultaneous analysis of molecular recognition, cell adhesion, and cell activation. Our applications will validate the capabilities of the instruments and extend our present knowledge of vascular traffic signals in novel ways to both protein and carbohydrate dependent adhesion. The technological capabilities that result will extend flow cytometry to ligand binding, high throughput cell screening, and automation of complex sample handling procedures.
Funding Period: 1999-05-01 - 2006-04-30
more information: NIH RePORT

Top Publications

  1. ncbi Dissociation of I domain and global conformational changes in LFA-1: refinement of small molecule-I domain structure-activity relationships
    Richard S Larson
    Department of Pathology, University of New Mexico, and Cancer Research and Treatment Center, Albuquerque, New Mexico 87131 5301, USA
    Biochemistry 44:4322-31. 2005
  2. ncbi Regulation of cell adhesion by affinity and conformational unbending of alpha4beta1 integrin
    Alexandre Chigaev
    Department of Pathology and Cancer Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
    J Immunol 178:6828-39. 2007
  3. pmc Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides
    Yang Wu
    Department of Pathology and Cancer Research Facility, University of New Mexico Health Sciences Center, University of New Mexico, Albuquerque, NM 87131, USA
    Anal Biochem 371:10-20. 2007

Scientific Experts

  • Richard Larson
  • Yang Wu
  • Tione Buranda
  • Larry A Sklar
  • Alexandre Chigaev
  • Gabriel P Lopez
  • Gordon J Zwartz
  • Peter C Simons
  • Anna Waller
  • James C Garrison
  • Eric R Prossnitz
  • William E McIntire

Detail Information

Publications3

  1. ncbi Dissociation of I domain and global conformational changes in LFA-1: refinement of small molecule-I domain structure-activity relationships
    Richard S Larson
    Department of Pathology, University of New Mexico, and Cancer Research and Treatment Center, Albuquerque, New Mexico 87131 5301, USA
    Biochemistry 44:4322-31. 2005
    ..DTT, in contrast, induces a conversion to the extended form of LFA-1 in the presence of BIRT-377 on live cells. The structure-activity studies suggest that BIRT-377 is a fully optimized inhibitor...
  2. ncbi Regulation of cell adhesion by affinity and conformational unbending of alpha4beta1 integrin
    Alexandre Chigaev
    Department of Pathology and Cancer Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
    J Immunol 178:6828-39. 2007
    ....
  3. pmc Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides
    Yang Wu
    Department of Pathology and Cancer Research Facility, University of New Mexico Health Sciences Center, University of New Mexico, Albuquerque, NM 87131, USA
    Anal Biochem 371:10-20. 2007
    ..These results are compatible with a cell activation model involving G protein conformational changes rather than disassembly of Galphabetagamma heterotrimer...