Tert butyl hydroperoxide tbhp

Whole-cell currents were elicited from injected oocytes and recorded to a desktop PC using the two-electrode voltage clamp technique as described previously [35] (link). Briefly, microelectrodes were fabricated from borosilicate glass capillaries using a micropipette puller and back-filled with 3 M KCl solutions. The WinWCP software (John Dempster, University of Strathclyde, Glasgow, UK) was used to control the voltage clamp amplifier (Oocyte Clamp, Warner Instruments, Hamden, CT). The data were digitized and low-pass filtered at various frequencies depending on the sampling rate. Tert-butyl hydroperoxide (tBHP, 70% from Acros Organics) and diamide (Sigma-Aldrich) solutions were prepared immediately before application and diluted to their nominal concentrations. Concentrations are described as “nominal” since the intracellular concentrations of these drugs cannot be determined precisely under our experimental conditions. When assayed with oocyte recordings, the dose-response curves of many compounds reportedly exhibit a rightward shift, and this effect has been proposed to be due to non-specific binding to the oocyte yolk and vitelline membranes [38] (link). Two (2) minutes were allowed between fluid exchanges.

For H₂O₂ treatments, H₂O₂ was diluted in PBS, then added directly to the media to get the final concentrations indicated in each experiment. The stock of H₂O₂ (Fisher Scientific AAL13235AP) was replaced monthly. For glucose oxidase (GO) treatments (Sigma, G7141-50KU), the desired concentration of the enzyme was made in sodium acetate (JTBaker, 3460-01) buffer (50 mM) and added directly to the media. For J14 (MedChemExpress, HY-135008), a stock of 10 mM was created in DMSO and added directly to the media for a final concentration of 20 μM. A stock concentration of 35 mM of Conoidin A (Cayman Chemical Item no. 15605) was made in DMSO. The stock was diluted in media and then added to cells to obtain the indicated final concentration. Tert-butyl hydroperoxide (TBHP, 70% solution in water) was obtained from Acros Organics and diluted in PBS. The TBHP PBS stock was then added directly to media to obtain the indicated final concentration. A stock solution of Menadione (Sigma Aldrich M5625) was made in 100% Ethanol, diluted in media, and then added to cells to obtain the final concentration indicated for each experiment.