Las x integrated imaging system

The cellular cholesterol content of fcwf-4 cells was evaluated by the Cholesterol Cell-Based Detection Assay Kit (Cayman chemical, USA) according to the manufacturer’s instructions. Briefly, fcwf-4 cells were grown on an 8-well Lab-Tek Chamber Slide (Thermo Fisher Scientific, Waltham, MA, USA). Semi-confluent fcwf-4 cell monolayers were cultured in medium containing compounds at 37 °C for 24 h. After fixing and staining, filipin III-binding cells were analyzed using a Leica DM4B microscope and LAS X integrated imaging system (Leica Microsystems, Wetzlar, Germany).

A cat was administered U18666A subcutaneously at 2.5 mg/kg. Peripheral blood was collected before and 24 h after administration. Heparinized blood was 2-fold diluted with PBS, and subjected to Ficoll-Hypaque (lymphoprep, Alere Technologies AS, Oslo, Norway) density gradient centrifugation at 800 g for 20 min. The PBMC layer was collected and washed three times. After cell counting, the PBMC was seeded to an 8-well Lab-Tek Chamber Slide (Thermo Fisher Scientific, Waltham, MA, USA) at density of 1 × 10⁵ cells/well. Slide was incubated at 37 °C for 2 h. After washing three times, adherent cells were fixed and stained. The cellular cholesterol was stained by the Cholesterol Cell Based Detection Assay Kit (Cayman chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. After filipin III staining, cells were stained by propidium iodide (PI, NACALAI TESQUE, INC., Kyoto, Japan) and analyzed using a Leica DM4B microscope with LAS X integrated imaging system (Leica Microsystems, Wetzlar, Germany).