I. Introduction:

Cholesterol is an essential structural component of animal cell membranes. It is required to maintain membrane structural integrity and fluidity.
胆固醇是动物细胞膜的重要结构成分。要求保持膜结构的完整性和流动性。
Cholesterol also serves as a precursor for the biosynthesis of several hormones, bile acid and vitamin D.
胆固醇也是多种激素、胆汁酸和维生素D生物合成的前体。
Plasma membrane contains majority of the cellular cholesterol (80-90%) whereas little cholesterol resides in endoplasmic reticulum and in mitochondrial membrane.
质膜含有大部分细胞胆固醇（80-90%），而少量胆固醇存在于内质网和线粒体膜中。
Transport of intracellular cholesterol within the cells to different compartments is through vesicular and non-vesicular pathways.
细胞内胆固醇在细胞内通过囊泡和非囊泡途径运输到不同的室腔。
Defects in these transport processes can alter cellular cholesterol metabolism resulting in pathological conditions.
这些转运过程中的缺陷可以改变细胞胆固醇代谢，从而导致病理状况。
Filipin III is widely used as a probe for sterol localization in membranes. Interaction with cholesterol alters Filipin absorption and fluorescence spectra.
Filipin Ⅲ是一种广泛应用于膜内固醇定位的探针。Filipin与胆固醇互作改变了Filipin吸收和荧光光谱。
This assay provides a simple, easy to perform, histochemical method of identification of unesterified cholesterol.
这种分析方法提供了一种简单，易于操作的组织化学方法来鉴定未酯化胆固醇。

II. Application:

Screen/study/characterize stimulators/inhibitors that affect cholesterol transport and localization
筛选/研究/鉴定影响胆固醇转运和定位的刺激物/抑制剂
VII. Cholesterol Detection Protocol:
This protocol is for a 96-well plate. Adjust the volume according to the plate size.

Cell Culture: Seed 2-3 x 104
cells/well in a 96-well plate in desired media. Grow cells overnight in 37°C incubator containing 5% CO2.
Next day, treat cells with compounds of interest in 100 µl media. As a control, we recommend treating cells with vehicle alone. For inhibitor control, treat cells with diluted Inhibitor (U-18666A, a cholesterol transport inhibitor). Grow cells for 48-72 hrs or for desired time period.
第二天，用100 µl培养基中感兴趣的化合物处理细胞。作为对照，我们建议单独用载体处理细胞。对于抑制剂控制，用稀释的抑制剂(U-18666A，胆固醇转运抑制剂)处理细胞。培养细胞48-72小时或所需的时间。
Note: Treatment with Inhibitor (U-18666A) will vary depending on the cell type. For HepG2 cell line we recommend using 1.25 μM.
注:使用抑制剂(U-18666A)的处理会根据细胞类型而有所不同。对于HepG2细胞系，我们建议使用1.25 μM。
Cell Staining: Remove culture media from wells.
细胞染色:从孔中去除培养基。
Wash cells with 100 µl Assay Buffer.
用100个测定缓冲液清洗细胞。
Add 50-100 µl of Fixative Solution and incubate for 10 min.
加入50-100µl的固定液孵育10分钟，
Wash cells with 100 µl Assay Buffer 2-3 times.
用100 µl 的缓冲液洗涤细胞2-3次。
Dilute Staining Dye 1:100 in Assay Buffer just before use and add 100 µl/well.
使用前将染色染料在缓冲液中稀释1:100，加入100个染色倍数l/well。
Incubate in dark for 30-60 min at 37°C.
37℃避光孵育30-60分钟。
Carefully remove the Assay Buffer containing dye using a pipette without disturbing the cells.
在不干扰细胞的情况下，用移液管小心地移走含染料的试验缓冲液。
Gently wash the cells 2-3 times with 100 µl Assay Buffer.
用100 mol / l试验缓冲液轻轻洗涤细胞2-3次。
Note: Cells can be stained directly with the Staining Dye without adding Fixative Solution (fixing of cells).
Detection: Examine cells using light and fluorescence microscope (Ex/Em = 340-380/385-470 nm). Acquire several images per well for analysis.
检测:使用光显微镜和荧光显微镜(Ex/Em = 340-380/385-470 nm)检测细胞。每口井采集几张图像进行分析。
Note: Since Staining Dye photobleaches very rapidly, we recommend analyzing samples immediately.
注:由于染色染料光漂白非常快，我们建议立即分析样品。