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Ildo Nicoletti, Roberta Mannucci Specimen: NIH-3T3 cells with hydrogen peroxide added Instrumentation: Olympus IMT-2 inverted microscope equipped with a Bio-Rad MRC 1024 Laser Scan Confocal Apparatus |
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Description: Under condition of stress, such as free radical excess or reactive oxygen generation, mitochondria undergo an autocatalytic collapse associated with the loss of the normal membrane potential. The transition is irreversible and is normally a prelude to cell death. The mitochondrial response to the hydrogen peroxide was investigated at the confocal microscope. At this purpose, NIH-3T3 cells were grown on a coverslip and stained with JC-1. The coverslip was then mounted on an Attofluor chamber (Molecular Probes) with 1 ml Krebs Ringer buffer and observed directly under confocal microscope. A single cell plane was chosen and the resulting image recorded at 5 min interval. The image shows normal mitochondria (elongated, red and green emission of JC-1) in a NIH-3T3 cell before the addition of hydrogen peroxide. |
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Description: The image shows the same focal plane 30 minutes after the addition of 0.02% hydrogen peroxide (HP). HP produced a fall in the red emission (due to the collapse in mitochondrial potential) alteration in mitochondrial structure (rounding and ballooning) and diffusion of green fluorescence into the cell cytosol. |
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