Technical Reference #1845

1845.

Caveolins sequester FA on the cytoplasmic leaflet of the plasma membrane; augment triglyceride formation; and protect cells from lipotoxicity Jeffrey R. Simard; Tova Meshulam; Biju K. Pillai; Micahel T. Kirber; Kellen Brunaldi; Su Xu; Paul F. Pilch; and James A. Hamilton, Boston University School of Medicine, Journal of Lipid Research, 51(1845), (2010)
Link To Paper

Keywords:
caveolae; caveolin-1; caveolin-3; cholesterol; lipid raft; transport; fatty acid

Materials & Methods:
For single-cell imaging experiments the desired amount of FPE stock was dried and resuspended in DMSO for addition to cells that were attached to the bottom of cover slips and grown as described above (4.2 M fi nal FPE concentration). In the case of BCECF cells were incubated in culture medium containing 2 mM BCECF-AM. After an incubation time of 30–40 min in the dark with gentle agitation the culture medium was replaced by MOPS buffer (pH 7.4) and cells were incubated for 40 min at 37°C prior to microscopic imaging. For imaging changes in FPE and BCECF fl uorescence in single cells a concentration of 20 M OA was added to 2 ml of MOPS buffer in a collagen-coated glass bottom culture dish from MatTek Corporation (Ashland MA) containing HEK293 cells. Cells were allowed to incubate with FA for several minutes before images were obtained with a two-photon laser scanning confocal microscope (excitation wavelength = 780 nm). Unlike the online fl uorescence measurements described above kinetic measurements with this approach are limited by the inability to mix the FA rapidly with the cells. Therefore detection of FA binding and transmembrane movement by each probe is limited by the time required for the FA to diffuse through the buffer and reach the cell surface.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
HEK293 cells