Protocol for hypoxanthine/xanthine oxidase assay
Stocks
o
100 mLs Potassium chloride (KCI) buffer: Make up in water
 120 mM KCl (MW 74.55)
 Powder: 0.8946 g
 10 mM HEPES (MW 238.3 g)
 Powder: 0.238.3
 1 mM EGTA (MW 380.4)
 1M Stock: 3.804 g in 10 mL NaOH
 0.1 mL of 1M solution
 pH 7.2
Reagents
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Dihydroethidium (DHE) (Sigma D7008)
o 10 mM stock
o 1 mg aliquots supplied
o Add 317 L DMSO
Hypoxanthine (MW 136.11)
o 1M stock: 1.3511 g in 10 mL KCl buffer
Xanthine oxidase (Sigma X7375) 154 mU/mL stock
o 196 l XO
o 804 l PBS
Catalase (Sigma C9322) 450 U/mL
o 10 mg/mL in 50 mM PPB
o 1 hr at 37 oC
o Dilute to 1 mg/mL prior to use in cold PPB
Superoxide dismutase (SOD) (Sigma S8160) 195 U/mL
o 1 mg/mL in 0.1M PPB (dilute above PPB by 10)
For 96 well plate: 200 L in each well
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2 L DHE (100 M/well)
2 L Hypoxanthine (1 mM)
6.5 L Xanthine oxidase (5 mU/mL)
22 L Catalase (50 U/mL)
2 L SOD
Make up to 200 L with KCl buffer
Shchepinova et al, 2017: MitoNeoH or MitoNeoD (10 mM) was incubated with 1 mM HX and 5
mU/mL XO, in the presence of 10 g/mL SOD and 50 U/mL catalase in KCl buffer at 37oC