Team:Caltech/Protocols/Measuring H2O2

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Latest revision as of 00:24, 30 October 2008

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Measuring H₂O₂

Note: Based on manufacturer's [http://www.piercenet.com/files/0526dh4.pdf instructions].

Spin down approximately 100 uL of cell culture
Measure peroxide concentration using a commercial [http://www.piercenet.com/Products/Browse.cfm?fldID=02020301 colorimetric assay] following the manufacturer's [http://www.piercenet.com/files/0526dh4.pdf instructions].
After incubating the reaction at room temperature for at least 20 minutes, measure the absorbance at 595 nm on a plate reader (Spectramax M2).
Calculate the concentration of hydrogen peroxide by reference to a standard curve (100uM, 33.3uM, 11.1 uM, 3.7 uM and 0.0 uM H2O2) made in the corresponding media.
- NB: Dilute culture supernatants as appropriate to bring measurements into the assay’s linear range.

An example of a typical standard curve generated by the assay. Error bars (1SD, n=3) are too small to be seen.

@@ Line 11: / Line 11: @@
 # Spin down approximately 100 uL of cell culture
 # Measure peroxide concentration using a commercial [http://www.piercenet.com/Products/Browse.cfm?fldID=02020301 colorimetric assay] following the manufacturer's [http://www.piercenet.com/files/0526dh4.pdf instructions].
-# After incubating the reaction at room temperature for at least 20 minutes, read on a plate reader (Spectramax M2) at 595nm.
+# After incubating the reaction at room temperature for at least 20 minutes, measure the absorbance at 595 nm on a plate reader (Spectramax M2).
 # Calculate the concentration of hydrogen peroxide by reference to a standard curve (100uM, 33.3uM, 11.1 uM, 3.7 uM and 0.0 uM H2O2) made in the corresponding media.
 #* NB: Dilute culture supernatants as appropriate to bring measurements into the assay’s linear range.