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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Is it possible to use the Amplex Red Reagent (Cat. No. A12222) with a colorimetric microplate reader and measure the optical density (OD)? What would be the ideal wavelength for OD measurements?
Yes it can be used for a colorimetric assay. In general, absorbance is less sensitive than fluorescence so the sensitivity of the colorimetric assay may be lower. You may need to empirically determine the detection limit and linear dynamic range for the colorimetric assay. Resorufin, the fluorescent product of Amplex Red, has an absorbance peak at 570 nm.
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I have ordered and tested your Amplex Red Reagent (Cat. Nos. A12222, A22177), but it is not reacting correctly. What could be the cause?
Either the horseradish peroxidase or other enzymes in the assay may not be active, the H2O2 may have degraded, or the sample could have some component that is consuming H2O2 or inactivating the enzymes required for the assay. We recommend that you check when the reagent was purchased and how it was stored prior to use; the reagent should not be used past the warranty period.
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Do you recommend using Amplex Red Reagent (Cat. Nos. A12222, A22177) in live cells?
Amplex Red is cell impermeant, but also its conversion to resorufin requires direct contact with the reaction of peroxidase with H2O2 (in solution, at the surface, or within cells). Within cells, peroxidases and H2O2 may not be sufficiently localized or active to provide enough signal.
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Can I use Amplex Red Reagent (Cat. Nos. A12222, A22177) in the presence of catalases?
Unless you are specifically analyzing catalase activity, as with the Amplex Red Catalase Assay Kit (Cat. No. A22180), the assay should be devoid of catalases. Catalases compete with the horseradish peroxidase for H2O2; the conversion of Amplex Red to resorufin requires the reaction of horseradish peroxidase with H2O2.
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