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.

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 <div style="font-size:18pt;">
-<font face="verdana" style="color:#CC3300">H<sub>2</sub>O<sub>2</sub> Production Assay</font></div>
+<font face="verdana" style="color:#CC3300">Measuring H<sub>2</sub>O<sub>2</sub></font></div>
 __NOTOC__
 <br>
-Approximately 100 uL of cell culture was spun down. A colorimetric assay was performed using the PeroXOquant™ Quantitative Peroxide Assay Kit (Pierce) as per the manufacture’s instructions in a 96 well plate. After incubating the reaction at room temperature for at least 20 minutes, it was read on a plate reader (Spectramax M2) at 595nm. The concentration of hydrogen peroxide was calculated by reference to a standard (100uM, 33.3uM, 11.1 uM, 3.7 uM and 0.0 uM H2O2) made in the corresponding media. Culture supernatants were diluted as appropriate to bring measurements into the assay’s linear range.
+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.
+[[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.]]
 }}