Evos fl color

Mitochondrial membrane potential (ΔΨm) was measured using the JC-1 assay kit (Beyotime Biotechnology, China) according to the manufacturer's instructions. After treatment, cells were incubated with JC-1 (2 μM final concentration) at 37°C in the dark for 30 min. Cells were then washed three times with buffer and observed using fluorescence microscopy (EVOS® FL Color, Life Technologies, Carlsbad, CA, USA). For further analysis, freshly isolated mitochondria were incubated with an equal volume of JC-1 staining solution (10 mg/mL) for 20 min at 37°C in the dark and rinsed twice with buffer. JC-1 fluorescence was measured using a microplate reader (Infinite M1000, Tecan). The green JC-1 signal was detected at excitation wavelengths of 490 nm and emission wavelengths of 530 nm. The red signal was detected at excitation wavelengths of 525 nm and emission wavelengths of 590 nm. The ratio of red and green fluorescence intensities indicated changes in the mitochondrial membrane potential.

DAPI staining was performed using MCF-7 cells that were cultured in a 12-well tissue culture grade plate (Nest, Wuxi, China) for 24 h. After incubation with IC₅₀ for 24 h, cells were washed in phosphate buffered saline (PBS) and fixed with ethanol for 15 min at room temperature. Cells after washing with PBS were stained with DAPI (2 μg/mL) and incubated in the dark for 30 min. The cells were then examined and imaged using a fluorescence microscope (EVOS^® FL Color, Life Technologies, Carlsbad, CA, USA).
For acridine orange/ethidium bromide assay, the amount of 2 µL of acridine orange/ethidium bromide (one part each of 3 mg/mL acridine orange and 3 mg/mL ethidium bromide in PBS) was mixed with 1 ml cell suspension in a 12-well plate. Cells were examined by EVOS^® imaging connected to a digital imaging system.

For analysis of the effects of GP-17 on ΔΨm, the change in ΔΨm was detected by JC-1 staining. HUVECs (10⁵ cells per well) were cultured in 24-well plates. After treatment, the cells were harvested and incubated with JC-1 (10 µM final concentration) at 37 °C in the dark for 30 min. The cells were immediately observed using fluorescence microscopy (EVOS^® FL Color, Life Technologies).
For further analysis of the fluorescence intensity, the treated cells were resuspended in fresh medium containing JC-1 (10 µM final concentration) and incubated at 37 °C in the dark for 30 min. The green JC-1 signal was measured at wavelengths of 485 nm for excitation and 535 nm for emission, the red signal at wavelengths of 540 nm for excitation and 590 nm for emission. The fluorescence intensity changes were analyzed using the automatic plate reader (Infinite M1000, Tecan).

The MPTP opening in H9c2 cardiomyocytes was measured via calcein AM staining using a living cell MPTP fluorescence detection kit (Genmed Scientifics Inc., Shanghai, China). After treatment, the cells were washed twice with reagent A and then incubated with an intermixture of reagent B and reagent C for 20 min at 37°C in the dark. After staining, cells were washed twice with reagent A, followed by image acquisition using fluorescence microscopy (EVOS® FL Color, Life Technologies). The fluorescence intensity of the mitochondria was determined using a microplate reader (Infinite M1000, Tecan) at an excitation wavelength of 488 nm and an emission wavelength of 505 nm.

The intracellular ROS production was monitored using a fluorescent probe DCFH-DA. After treatment, cells were incubated with 10 μM DCFH-DA for 25 min at 37 °C and then washed thrice with phosphate-buffered saline (PBS) buffer. Finally, cellular morphology and fluorescence distributions were observed using a fluorescence microscopy (EVOS^® FL Color, Life Technologies).
After treatment, cells were harvested by 0.25% trypsin and washed by PBS buffer. And then cells were centrifuged and incubated with 5-(and-6)-carboxy-2′, 7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) in the dark at 37 °C for 30 min. The fluorescence was analyzed by a flow cytometry (BD, Biosciences, CA, USA).

A live/dead cell viability assay (Thermo
Fisher Scientific, UK) was utilized to determine the live and dead
cell population after the ALA, QD, and ALA-loaded QD treatment before
and after irradiation. HT29 and SW480 cells were seeded following
the procedure explained for the ROS assay. After irradiation at 420
nm for 5 min, the cells were stained with the live/dead assay based
on the protocol that is provided by the manufacturer. Finally, images
were collected using an inverted fluorescence microscope (EVOS FL
color, Life Technologies, Thermo Fisher Scientific, UK).

The change in mitochondrial membrane potential was detected by JC-1 staining. After treatment, the cells were incubated with JC-1 (2 μM final concentration) at 37°C in the dark for 30 min. Then the cells were washed three times with PBS and observed using fluorescence microscopy (EVOS^® FL Color, Life Technologies). For further analysis of the fluorescence intensity, the treated cells were harvested and incubated with JC-1 at 37°C in the dark for 30 min. The fluorescence intensity changes were analyzed using a microplate reader (TECAN Infinite M1000, Austria). The green JC-1 signal was detected at excitation wavelengths of 514 nm and emission wavelengths of 529 nm, the red signal was detected at excitation wavelengths of 585 nm and emission wavelengths of 590 nm.