Mitochondrial membrane potential in L. vannamei hemocytes

Description

Mitochondria are an organelles with multiple functions related to cell survival, and changes in mitochondrial activity can affect the cell’s health. Mitochondrial membrane permeabilization (MMP) is a critical event that lead to mitochondrial mediated cell death. MMP triggers several subsequent events, such as the release of pro-apoptotic molecules, the disruption of cellular energy metabolism and the loss of mitochondrial membrane potential. In shrimp hemocyte primary cell culture, the mitochondrial membrane potential can be monitored by staining with JC-1 dye (5, 5', 6, 6' -tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide; 5 µg/ml in 2x L-15 medium). Cells stained with this dye present different colors depending on whether the cells have high or low mitochondrial membrane potential.

Brief protocol

Exposed the seeded shrimp hemocyte monolayers to JC-1 dye (5, 5', 6, 6' -tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide; 5 µg/ml in 2x L-15 medium) for 30 min. After counterstaining the nucleus with DAPI (4'-6-Diamidino-2-phenylindole) for 5 min, the cells were washed twice again and fluorescent signals were viewed with a microscope. In each treatment, 300 ~ 1,500 hemocytes were investeaged. Data were subjected to unpaired Student’s t-tests. P values of less than 0.05 were considered to be a statistically significant difference. The positive control in this experiment was the UV-light treatment.