In total, 1 µL of an A. fumigatus spore suspension (107/mL) was inoculated on PDA in the presence or absence of 1 mM farnesol plus 0.1 mM cPTIO (Dojindo, Kumamoto, Japan) and 0.1 mM tocopherol (NACALAI TESQUE, Inc.). After incubation at 37 °C for 3 days, the diameters of colonies were measured. The IC50 values were calculated using KaleidaGraph (Synergy Software, Reading, PA).
Cptio
Manufactured by Dojindo Laboratories
Sourced in Japan
CPTIO is a water-soluble cell-permeable spin probe for EPR (Electron Paramagnetic Resonance) spectroscopy. It can be used to measure the redox status and oxidative stress within living cells.
Automatically generated - may contain errors
2 protocols using cptio
1
Antifungal Effects of Farnesol and Cptio
2
AMPK Activity Measurement using ADP-Glo Assay
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Caspase-3 activity was determined using the NucView488 and RedDot2 apoptosis and necrosis kit (Biotium), according to the manufacturer's instructions. Dead cells were stained with RedDot2 or DRAQ7 (BioStatus, Shepshed, UK). HeLa cells were incubated with the vehicle, 0.2 mM cPTIO (Dojindo), or 4 mM L-NAME (Nacalai Tesque) for 1 h before L-c414 was added. The data were analyzed by using the Accuri C6.
In vitro kinase assay For the assay, 0.5 ng/ml full-length recombinant human AMPKa1b1g1 (SignalChem, Richmond, BC, Canada) was incubated for 60 min with 0.2 mg/ml of SAMS peptide (Abcam, Cambridge, UK) and 150 mM ATP in the kinase assay buffer [2.5 mM MOPS (pH 7.2), 1.25 mM b-glycerol-phosphate, 2.5 mM MgCl 2 , 0.5 mM EGTA, 0.2 mM EDTA, 0.25 mM DTT, 0.01 % (w/v) Brij-35, 0.25 % (w/v) bovine serum albumin (BSA), and 0.125 % (w/v) Tween 80] at 25 C. ATP hydrolysis resulting from the kinase reactions was detected using the ADP-Glo kinase assay kit (Promega).
In vitro kinase assay For the assay, 0.5 ng/ml full-length recombinant human AMPKa1b1g1 (SignalChem, Richmond, BC, Canada) was incubated for 60 min with 0.2 mg/ml of SAMS peptide (Abcam, Cambridge, UK) and 150 mM ATP in the kinase assay buffer [2.5 mM MOPS (pH 7.2), 1.25 mM b-glycerol-phosphate, 2.5 mM MgCl 2 , 0.5 mM EGTA, 0.2 mM EDTA, 0.25 mM DTT, 0.01 % (w/v) Brij-35, 0.25 % (w/v) bovine serum albumin (BSA), and 0.125 % (w/v) Tween 80] at 25 C. ATP hydrolysis resulting from the kinase reactions was detected using the ADP-Glo kinase assay kit (Promega).
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