Overview

  • Product name

    Cholesterol Assay Kit (Cell-Based)
    See all Cholesterol kits
  • Detection method

    Fluorescent
  • Sample type

    Adherent cells
  • Assay type

    Cell-based
  • Assay time

    1h 15m
  • Species reactivity

    Reacts with: Mammals, Other species
  • Product overview

    Cholesterol Assay Kit (Cell-Based) (ab133116) includes filipin III, fixative, and wash buffer in a ready to use format. It provides a simple fluorometric method to study mechanisms and biological factors that regulate cholesterol metabolism or movement within cells. A cholesterol trafficking inhibitor, U-18666A, is included as a positive control.


    Cholesterol assay protocol summary:
    - remove medium from cells
    - fix with fixative solution for 10 min
    - wash with wash buffer 3 times for 5 min
    - add Filipin III solution and incubate for 30-60 min
    - wash twice for 5 min
    - analyze with fluorescent microscope

  • Notes

    Cholesterol is both an important structural component of cell membranes and an early intermediate in hormone and bile acid biosynthesis. Cholesterol is not uniformly distributed among cellular membranes /  organelle membranes, but rather there are structurally and kinetically distinct cholesterol rich and poor domains.

    Filipin III is the predominant isomer of filipin, the collective name given to four isomeric polyene macrolides isolated from cultures of S. filipinensis. Filipin is widely used as a probe for sterol location in biological membranes.

    Interaction with cholesterol alters the filipin absorption and fluorescence spectra allowing visualization with a fluorescence microscope capable of excitation at 340-380 nm and emission at 385-470 nm. Filipin’s ease of use makes it a convenient tool for the histochemical identification of unesterified cholesterol both in vitro and in vivo.

    Other cholesterol assay kits include:
    HDL and LDL/VLDL Cholesterol assay kit ab65390 
    Cholesterol/Cholesterol Ester assay kit ab65359 
    Cholesterol Efflux assay kit ab196985 
    Cholesterol Uptake assay kit ab236212

  • Platform

    Fluorescence microscope

Properties

Images

  • HepG2 cells in response to 2 µM U-18666A.
    HepG2 cells were seeded in a 96-well plate at a density of 2.4 x 104 cells/well and cultured overnight. The next day, cells were treated with 2 µM U-18666A for 48 hours. U-18666A treatment for 48 hours induces intracellular accumulation.

  • HepG2 cells in response to 1 µM U-18666A.
    HepG2 cells were seeded in a 96-well plate at a density of 2.4 x 104 cells/well and cultured overnight. The next day, cells were treated with 1 µM U-18666A for 48 hours. U-18666A treatment for 48 hours induces intracellular accumulation.

     

  • HepG2 cells in response to 0.5 µM U-18666A.
    HepG2 cells were seeded in a 96-well plate at a density of 2.4 x 104 cells/well and cultured overnight. The next day, cells were treated with 0.5 µM U-18666A for 48 hours. U-18666A treatment for 48 hours induces intracellular accumulation.

  • HepG2 cells in response to 0.25 µM U-18666A.
    HepG2 cells were seeded in a 96-well plate at a density of 2.4 x 104 cells/well and cultured overnight. The next day, cells were treated with 0.25 µM U-18666A for 48 hours. U-18666A treatment for 48 hours induces intracellular accumulation.

     

  • Accumulation of cholesterol inside HepG2 cells - Control.
    HepG2 cells were seeded in a 96-well plate at a density of 2.4 x 104 cells/well and cultured overnight. The next day, cells were treated with DMSO (vehicle) for 48 hours. Cells demonstrate that majority of cholesterol is localized on the plasma membrane.

  • Accumulation of cholesterol inside HepG2 cells in response to 1.25 µM U-18666A.
    HepG2 cells were seeded in a 96-well plate at a density of 3 x 104 cells/well and cultured overnight. The next day, cells were treated with DMSO (vehicle) or 1.25 µM U-18666A for 48 hours. Panel A: Cells treated with DMSO alone demonstrate that majority of cholesterol is localized on the plasma membrane. Panel B: U-18666A treatment for 48 hours induces intracellular accumulation.

Protocols

References

This product has been referenced in:

See all 9 Publications for this product

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Cells were seeded in 96-well plate and cultured for 36h. Experiment has been performed by Julie Gamart, PhD.

Dr. Julie Gamart

Verified customer

Submitted Apr 17 2018

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