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 timeand 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°C efficiently resolved detergent solubility or resistance of

Age-Related Defects in Moesin/Ezrin Cytoskeletal Signals in Mouse CD4 T Cells

“…We and others have shown that some membrane-associated proteins can be resistant to Brij-58 detergent extraction under specific conditions (28,29,37,38). We evaluated the effects of Brij extraction on ERM protein distribution in freshly isolated CD4 T cells.…”

E‐LDL and Ox‐LDL differentially regulate ceramide and cholesterol raft microdomains in human Macrophages

“…They are characterized by the enrichment of cholesterol, sphingolipids, and saturated phospholipids, and, in addition, by their insolubility in different detergents such as Triton X-100 and Lubrol WX (4). Rafts are thought to function as platforms for the dynamic association of signaling molecules.…”

“…Mildly oxidative modification of LDL was achieved by dialyzing purified LDL fractions (1 mg protein/ml) against 5 lM CuSO 4 for 36 h, and afterwards, the Ox-LDL was sterile filtered. ), fixed in 4% paraformaldehyde for 15 min, quenched with 50 mM NH 4 Cl/glycin for 15 min, and then blocked with PBS/0.5% BSA for 15 min at RT. Afterwards, cells were incubated with anti-ceramide mAB 15B4 for 3 h at 4°C and then labeled with goat anti mouse-Cy5 (Abcam, Cambridge, United Kingdom) secondary antibody for 1 h at 4°C, followed by incubation with theta-toxin-FITC for 20 min at 4°C (KFB, Regensburg, Germany) for cholesterol staining.…”

The effects of detergents on the polymerization properties of actin

“…This raises the question, how the detergents influencing actin polymerization in vitro act on DRMs in intact cells. This approach is appealing, since recently, a flow cytometric method was developed for DRM isolation, called FCDR assay (29)(30)(31)(32)(33)(34). In these works, raft-associated proteins labeled by fluorescently tagged antibodies remained cell associated, after detergent solubilization of the whole cells.…”

“…Lipid rafts involved in cell signaling or membrane and protein trafficking are plasma membrane lipid heterogeneities containing elevated amounts of sphingolipids, cholesterol, GPI-anchored and other particular ''raft'' proteins (23)(24)(25)(26)(27)(28). The methodologically defined detergent-resistant membranes (DRMs), which considered to show comparable chemical properties to rafts, are identified applying the same TX100, Brij and CHAPS detergents and used in recently developed cytometric DRM measurements [flow cytometric detergent resistance (FCDR) assay] (29)(30)(31)(32)(33)(34).…”