Dear collegues,
Recently I've faced a strange problem using tert-butyl hydroperoxide for induction of oxidative stress and cell death. I've worked with neuroblastoma SH-SY5Y cell line. My aim was to find a suitable concentration of TBHP for 24 h incubation. Concentrations were: 10, 20, 50, 100, 200 and 500 μM. Medium: DMEM/f12 (10% BCS, 25 µg/mL gentamicin). In 96-well plate (Costar) 104 cells were put and medium till 100 μL of final volume. In 48-well plate (Eppendorf) 3x104 cell were put and medium till 200 μL of final volume. Incubation for 24 h under 37 C and 5% CO2. I've observed a strange situation. After incubation in all wells (both for 48 and 96 plate) including such with intact cells almost total cell death was found independently of TBHP concentration. But after incubation in plate with only intact cells and no TBHP there was a fine viability not less than 90%. Previously I've worked in the same conditions but with PBMCs and have no problems. Can some possibly explain that strange phenomenon? Thank you.
Dear Innokentiy
I have the feeling that your concentrations of butyl hydroperoxide are very high. You should start with much lower concentrations.
Another point you should check is the pH of the solution.
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