To investigate the MMP, the fluorescent JC-1 dye was used by following Saeed et al’s protocol.16 (link) There was a decrease in red/green fluorescence intensity ratio, an indication of mitochondrial depolarization by a fluorescence emission shift from green (529 nm) to red (590 nm). Healthy cells are in red while apoptotic cells are in green.
Cells were grown in six-well plates (1.5×105 cells/well) and treated with MTX (15, 25, and 50 µM). After 24 hours incubation, 5 mM JC-1 stain was added to each well and incubated at room temperature for 30 minutes, and images were visualized by fluorescence microscope (Nikon Eclipse 80i with Nikon DS-Ri1 12.7-megapixel camera; Nikon Corporation).
The quantitative result was examined by using a fluorescence microplate reader (FLUO star Omega; BMG Labtech) at 529 nm excitation and 590 nm emission.
Cells were grown in six-well plates (1.5×105 cells/well) and treated with MTX (15, 25, and 50 µM). After 24 hours incubation, 5 mM JC-1 stain was added to each well and incubated at room temperature for 30 minutes, and images were visualized by fluorescence microscope (Nikon Eclipse 80i with Nikon DS-Ri1 12.7-megapixel camera; Nikon Corporation).
The quantitative result was examined by using a fluorescence microplate reader (FLUO star Omega; BMG Labtech) at 529 nm excitation and 590 nm emission.
