Invitrogen™ Amplex™ Red Cholesterol Assay Kit

Catalog No.	Quantity
A12216	500 Assays

Catalog No. A12216 Supplier Invitrogen™ Supplier No. A12216

Description

Includes

Amplex Red reagent, DMSO, Horseradish peroxidase, Hydrogen peroxide, Cholesterol esterase, Cholesterol oxidase, Cholesterol reference standard, Resorufin fluorescence reference standard, 5X Reaction buffer

Detects both free cholesterol and cholesteryl esters

The Amplex™; Red Cholesterol Assay Kit provides a sensitive, rapid, and simple fluorometric method for detecting very low concentrations of cholesterol using a fluorescence microplate reader or fluorometer. The assay measures both free cholesterol and cholesteryl esters.

• Detects as little as 200 nM (80 ng/ml) cholesterol
• Helps accurately measure cholesterol content of 0.01 μl of human serum
• Detects both free cholesterol and cholesteryl esters
• Allows for multiple time point measurements
• Designed for minimal autofluorescence interference

The Amplex Red Cholesterol Assay kit contains these reagents and buffers:

• Amplex Red reagent
• DMSO
• Horseradish peroxidase
• Hydrogen peroxide
• Cholesterol esterase
• Cholesterol oxidase
• Cholesterol reference standard
• Resorufin fluorescence reference standard
• 5X Reaction buffer

Cholesterol Measurement is Simple and Fast

Performing the Amplex™ Red cholesterol assay is quick and easy, requiring only a 30-minute incubation after addition of reagents with no separation steps required. The 100 μl assay volume is designed for 96-well plates; fluorescence can be measured with a microplate reader or fluorometer. Since the assay is continuous, once the Amplex™ Red reagent is added, multiple time-point measurements can be taken. The kit is complete with all necessary reagents except deionized water.

Helps Accurately Detect Very Low Cholesterol Concentrations

The Amplex™ Red cholesterol assay utilizes enzyme-coupled reactions to produce highly fluorescent resorufin for quantitation. Resorufin is produced by the reaction of the Amplex™ Red reagent with H₂O₂ produced from the cholesterol oxidase-catalyzed oxidation of cholesterol. Because a large proportion of cholesterol in blood is in the form of cholesteryl esters, cholesterol esterase is used to produce free cholesterol from cholesterol esters. Performing the assay in the presence and absence of cholesterol esterase potentially allows determination of the fractions of cholesterol in each form.

This highly sensitive method allows the Amplex™ Red Cholesterol Assay kit to detect concentrations of cholesterol as low as 200 nM and measure the cholesterol content of 0.01 μl of human serum. Because the absorbance and emission maxima of resorufin are 571 nm and 585 nm respectively, autofluorescence in most biological samples does not interfere with the assay.

Use Amplex™ Red Assays for a Broad Range of Investigations

A wide variety of validated Invitrogen Amplex™ Red assays are available for studying cell signaling and lipids, neurobiology, inflammation and immune function, and metabolism. We also offer Amplex™ UltraRed (Cat. No. A36006), a second-generation reagent providing greater sensitivity and brighter fluorescence and the Amplex™ Red/UltraRed stop reagent (Cat. No. A33855). The Amplex™ Red/UltraRed stop reagent provides convenience and control by allowing the fluorescence signal-generating reaction to be terminated at a user-determined time point. After addition of the stop reagent, the fluorescence signal remains stable for at least 3 hours. Custom assay design and packaging are also available.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Order Info

Shipping Condition: Room temperature

Specifications

• Color: Red
• Content And Storage: Store in freezer -5°C to -30°C and protect from light.
• Detection Method: Fluorescence
• For Use With (Application): Enzyme and Metabolic Activity
• For Use With (Equipment): Microplate Reader
• Product Type: Amplex Red Assay Kit
• Dye Type: Amplex Red
• Format: 96-well plate
• Product Line: Amplex
• Quantity: 500 Assays
• Shipping Condition: Room Temperature
• Excitation Wavelength: 571 nm
• Emission Wavelength: 585 nm

Frequently Asked Questions (FAQs)

I'm using an Amplex Red kit, the reagent changes color to pink almost immediately in my own Krebs-Ringer buffer but not in HBSS. Why is this?

The components of Krebs-Ringer buffer (salts) should not cause oxidation of the Amplex reagent (which, in the presence of peroxidase and H₂O₂ oxidizes to resorufin, which is pink in color and fluorescent). Try water alone (the water used to make the Krebs-Ringer buffer). Since Hank's Buffered Saline Solution is typically purchased rather than made in the lab, it likely would not have the same contaminant. Another option is to degas the buffer prior to use to removed dissolved oxygen radicals.

For the lipid extraction for use with the Amplex Red Cholesterol Assay Kit (Cat. No. A12216), can the extraction be done in plastic tubes (Falcon tubes, Eppendorf tubes) or is glassware required?

You can use plastic tubes for the extraction of lipids if the plastic material is compatible with the organic solvents used in the extraction.

In order to determine the cholesterol content of my cells with the Amplex Red Cholesterol Assay Kit (Cat. No. A12216), do I need lysed cells or the extracted lipidome of the cell?

You need to extract the cholesterol from live or lysed cells. The following detergent‐free cholesterol extraction protocol may be of interest to you:

1. Add 200 µl or less of live cells or lysed cells into 200 µl chloroform‐methanol (v/v 2:1) or 200 µl hexane‐isopropanol (v/v 3:2).
2. Centrifuge for 5‐10 min at 14,000 rpm in a microcentrifuge.
3. Transfer the organic phase to a clean tube and vacuum dry.
4. Redissolve the dried lipids/cholesterol into 1X concentration of Component E, the reaction buffer.

Note: Use a volume of 1X reaction buffer sufficient to resolubilize the lipids/cholesterol. As a general guideline, one may use a volume of 1X reaction buffer equivalent to the original volume of cells or less.
Extraction method originated from the following publications:

1. Folch, J., Ascoli, I., Lees, M., Meath, J. A. & LeBaron, F. N. (1951) J. Biol. Chem. 191, 833-841
2. Folch, J., Lees, M. & Sloane-Stanley, G. H. (1957) J. Biol. Chem. 226, 497-509

Can I use Amplex Red Cholesterol Assay Kit (Cat. No. A12216) with cell or tissue samples?

Yes. The Amplex Red Cholesterol Assay Kit (Cat. No. A12216) can be used with cell or tissue samples. This assay requires samples containing cholesterol, free of any chemical or cellular components that may interfere with the activity of the enzymes in the assay or the performance of the dye. Below is a detergent/surfactant-free lysis/extraction method.

1. Homogenize 1 x 10⁶ cells or ~10 mg tissue into 200 µL chloroform-methanol (v/v 2:1) or 200 µL hexane-isopropanol (v/v 3:2).

Note: These solvents will cause the cells to disrupt immediately upon contact, but the homogenization (vortexing, sonicating, or mechanical homogenizers, etc.) helps to guarantee cell contact with the solvent.

2. Centrifuge for 5-10 min at 14,000 rpm in a microcentrifuge.

3. Transfer the organic phase to a clean tube and vacuum dry.

4. Redissolve the dried lipids/cholesterol into 1X concentration of Component E, the reaction buffer.

Note: Use enough volume of 1X reaction buffer sufficient to resolubilize the lipids/cholesterol. As a general guideline, you may use a volume of 1X reaction buffer equivalent to the original volume of cells or less.

Extraction method originated from:

Folch, J., Ascoli, I., Lees, M., Meath, J. A. & LeBaron, F. N. (1951) J. Biol. Chem. 191, 833-841

Folch, J., Lees, M. & Sloane-Stanley, G. H. (1957) J. Biol. Chem. 226, 497-509

Can Amplex Red Assays be performed using cell lysates?

This is not recommended. The presence of endogenous proteases can complicate the assay by degrading the horseradish peroxidase (HRP). Endogenous peroxidases and antioxidants can modify the H₂O₂ required for the reaction, competing with HRP (and catalase) for the substrate.

The Amplex Red Assays are best performed with either purified enzymes or extracted H₂O₂ in a defined buffer system, extracellular solutions or body fluids (media, serum, etc.) that do not exhibit high levels of endogenous protease or oxidase activity and do not contain antioxidants.

For Research Use Only. Not for use in diagnostic procedures.