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Team:Caltech/Protocols/Measuring H2O2
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# Spin down approximately 100 uL of cell culture | # Spin down approximately 100 uL of cell culture | ||
| - | # Measure peroxide concentration using a [http://www.piercenet.com/Products/Browse.cfm?fldID=02020301 colorimetric assay] | + | # 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, | + | # 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. | # 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. | #* NB: Dilute culture supernatants as appropriate to bring measurements into the assay’s linear range. | ||
| - | [[Image:H2O2 standards example.png|thumb|center|400px|An example of a typical standard curve generated by the assay. Error bars (1SD n=3) are too small to be seen.]] | + | [[Image:H2O2 standards example.png|thumb|center|400px|An example of a typical standard curve generated by the assay. Error bars (1SD, n=3) are too small to be seen.]] |
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Latest revision as of 00:24, 30 October 2008
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Measuring H2O2
Note: Based on manufacturer's instructions.
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