title: Cholesterol sensitivity of detergent resistance: a rapid flow cytometric test for detecting constitutive or induced raft association of membrane proteins.
abstract: BACKGROUND
  Lipid rafts are cholesterol- and glycosphingolipid-rich microdomains in the cellular plasma membranes that play critical roles in compartmentalization (concentration, coupling, and isolation) of receptors and signal molecules. Therefore, detecting constitutive or induced raft associations of such proteins is of central interest in cell biology. This has mostly been done with time- and cell-consuming immunobiochemical techniques affected by several sources of artifacts. A flow cytometric analysis of immunocytochemical staining under differential circumstances of detergent treatment offers a new alternative to this method.
  
  
  METHODS
  Membrane microdomains are resistant to nonionic detergents due to extensive, strong interactions between their molecular constituents. We used this feature to develop a rapid flow cytometric assay of differential detergent resistance based on immunocytochemical labeling of extracellular domain epitopes in membrane proteins. Data evaluation is based on comparative detection of their detergent solubility without and with cholesterol depletion of cell membranes, resolved by moderate concentrations of nonionic detergents.
  
  
  RESULTS
  Nonionic detergents Triton X-100 and Nonidet-40 (0.05-0.1%) in cold or Brij-98 (0.1-0.5%) at 37 degrees C efficiently resolved detergent solubility or resistance of many lymphocyte cell surface proteins. Kinetic data revealed that a short (5-10 min) detergent treatment is sufficient for this assay. Comparison of detergent solubility in untreated and cholesterol-depleted cells differentiated membrane proteins associated with or excluded from raft microdomains, respectively. Confocal microscopy showed that this mild detergent treatment leaves the cytoskeleton of the cells intact, with a detectable expression of raft marker detergent-resistant proteins attached to it. An induced association with rafts of immunoglobulin E receptors upon antigen cross-linking was also easily detectable in rat mast cells by this approach.
  
  
  CONCLUSIONS
  A protocol is proposed for a rapid (5-10 min) test of detergent resistance of membrane proteins in cells. The approach requires only a small amount of cells (10(4)/sample) and offers a good resolution of detergent solubility or resistance of membrane proteins, also in terms of the underlying mechanisms, with an advantage of applicability for all conventional bench-top flow cytometers.
authors.affiliation: Department of Immunology, Eotvos Lorand University, Budapest, Hungary.
authors.forename: Imre
authors.initials: I
authors.lastname: Gombos
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Bacsó
authors.forename: Cynthia
authors.initials: C
authors.lastname: Detre
authors.forename: Henrietta
authors.initials: H
authors.lastname: Nagy
authors.forename: Katalin
authors.initials: K
authors.lastname: Goda
authors.forename: Márton
authors.initials: M
authors.lastname: Andrásfalvy
authors.forename: Gábor
authors.initials: G
authors.lastname: Szabó
authors.forename: János
authors.initials: J
authors.lastname: Matkó
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology*; D004351:Drug Resistance; D000939:Epitopes / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008214:Lymphocytes / Q000378:metabolism; D008407:Mast Cells / Q000166:cytology; D021962:Membrane Microdomains / Q000737:chemistry*; D018613:Microscopy, Confocal; D008954:Models, Biological; D017830:Octoxynol; D010641:Phenotype; D011092:Polyethylene Glycols / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D051381:Rats; D017455:Receptors, IgE / Q000737:chemistry; D015398:Signal Transduction; D013696:Temperature; D013997:Time Factors
chemical_list: D003432:Cross-Linking Reagents; D003902:Detergents; D000939:Epitopes; D017455:Receptors, IgE; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D002784:Cholesterol