3). In contrast, a 10-min exposure to hydrophilic water-soluble NBD-2-deoxyglucose resulted in labeling of the entire population of fat cells in the explant (Fig. 1B). Dead cells did not accumulate a significant amount of Bodipy-C12, indicating selleck screening library that FA uptake requires membrane integrity (Figs. 1D and and5).5). Adipose tissue comprised of adipocytes with diameters >80�C100 ��m displayed a very different pattern of fluorescence; Bodipy-C12 staining was weak and diffuse, and dead and live fat cells accumulated similar amounts of fluorescence regardless of the presence of insulin (Fig. 1E). Fig. 3. Diffusion of Bodipy-C12 in adipose tissue. A: a sample of adipose tissue that was insulin resistant and transported Bodipy-C12 by diffusion was incubated with Bodipy-C12 and the fluorescent quencher, as described in materials and methods.

Time-lapse microscopy … Fig. 5. Active FA transport in adipose tissue. Insulin-stimulated retroperitoneal fat explants were incubated in medium alone (A and B) or in the presence of 100 ��M lipid mixture (C and D) prior to Bodipy-C12 addition. Nuclei of dead cells were visualized … Imaging through adipose tissue potentially imposes significant technical problems in that large lipid droplets scatter fluorescent light and create uneven illumination and light collection. Additionally, uneven diffusion of fluorescent dye in fat tissue may contribute to variability of results. To address the light-scattering problem, we analyzed three-dimensional images of the first layer of large insulin-nonresponsive adipocytes that showed a diffuse Bodipy-C12 pattern (Figs.

1E and and2A).2A). Vertical sections through the stacks showed that images were not severely distorted, the shape of adipocytes appeared principally circular, and fluorescence intensity did not vary with distance from the coverslip (Fig. 2, A and B). These experiments indicate that, under the imaging conditions used, spherical aberrations and scattering of fluorescence light from the outer cell layer of fat explants are negligible. Fig. 2. The effect of light scattering on Bodipy-C12 fluorescence in adipose tissue. Adipose tissue containing large fat cells (as in Fig. 1E) was labeled with Bodipy-C12. A: a closeup XYZ image of an outer layer of adipose tissue explant. The 148-��m-thick … Figure 3 illustrates the diffusion of Bodipy-C12 fluorescence in an explant containing large adipocytes.

There was an apparent delay in the accumulation of fluorescence, possibly because Bodipy-C12 must partition in and cross the lipid bilayer and subsequently accumulate in sufficient quantities to give a measurable intracellular AV-951 signal. At 600 s, Bodipy-C12 preferentially accumulated in the outer layer of the fat explant, with little labeling detected in deeper cell layers (Fig. 3, A and C).

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