Floid microscope

The mitochondrial membrane potential (MMP) was measured by staining astrocytes with JC-1 (5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazolyl-carbocyanine iodide) according to the method of Korenic et al. [51 (link)].
JC-1 is a ljipophilic and cationic dye that exhibits a fluorescence emission shift upon aggregation from 530 nm (green monomer) to 590 nm (red “J-aggregates’’ monomer). In healthy cells with high MMP, JC-1enters the mitochondrial matrix in a potential-dependent manner and forms aggregates.
Cells were seeded into groups as previously described. After treatment, cells were stained with 2.5 mg/mL JC-1 at 37^ºC for 15 min. Thereafter, cells were rinsed three times with PBS and the dye was allowed to equilibrate at room temperature for another 10 min before imaging using an Invitrogen Floid microscope (Fisher Scientific, Sweden). Stained polarized mitochondria were detected with red fluorescence while the loss of mitochondrial integrity was detected with green fluorescence. The fluorescence was read on a plate reader (Infinite 200Pro Tecan, Switzerland) with an excitation wavelength of 485 nm and the absorbance at 530 nm and 590 nm. The JC-1 fluorescence values were first normalized separately to their respective controls for red and green signals before the relative MMP was calculated.

The live/dead assay is used for assessing cell viability/death and was carried out using a live/dead viability/cytotoxicity assay kit for mammalian cells (Invitrogen Detection Technologies, Thermofisher, UK) according to the manufacturer’s instructions. This method is used for assessing cell viability/death in live cell culture of cells. It uses two components, namely; Calcein AM (CaAM), which is component A and Ethidium homodimer-1 (EthD-1), which is component B. Dead cells are characterized by intense fluorescence at over 600 nm and little fluorescence around 530 nm.
Astrocytes seeded in an 8-well chamber slide were allowed to adhere and grow to confluence for 14 h at 37 °C in a CO₂ incubator. Thereafter, cells were placed in a normoxic or severe hypoxic condition for 3 and 6 h. After 3 and 6 h, 100 μL of EthD-1 (1 μL/mL) and CaAM (0.2 μL/mL) were separately prepared in cell media (MEM), added to the cells, and then, incubated at 37^ºC for 10 min. Fluorescent images of the cells were taken at excitation/emission(ex/em) wavelength of 495 nm/515 nm for the green filter (live cells) and ex/em wavelength of 495 nm/635 nm for the red filter (dead cells) using an Invitrogen Floid microscope (Fisher Scientific, Sweden). Percentage cell death was calculated to get the number of dead cells.