Amplex™ Red Cholesterol Assay Kit - FAQs

View additional product information for Amplex™ Red Cholesterol Assay Kit - FAQs (A12216)

5 product FAQs found

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.

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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.

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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


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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

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.