Amplex™ Red Cholesterol Assay Kit

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.

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

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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.

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

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

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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.

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