Mitotracker cmx red

Mitochondrial mass estimation was performed with MitoTracker Green, while both MitoTracker Red and JC-1 were used to assess mitochondrial activity. MitoTracker assay was performed for 30 min in culture condition with 50 nM MitoTracker Red CMX (Thermo Fisher Scientific), 50 nM MitoTracker Green (Thermo Fisher Scientific), and Hoechst 33342 (Thermo Fisher Scientific). Then, cells were washed in PBS and glass coverslips were mounted on glass slides with a 90% (v/v) glycerol/PBS solution. MitoProbeTM JC-1 assay (Thermo Fisher Scientific) was performed for 30 min in culture condition with 2 μM JC-1, and Hoechst 33342 (Thermo Fisher Scientific). The mitochondrial membrane potential disrupter CCCP (Thermo Fisher Scientific) was used at 50 μM as a control.

Mitotracker CMX Red(Invitrogen) was used to determine the mitochondrial membrane
potential (Ψ_mit) of treated parasites. After 6, 16, 24, and 48 hours
treatment, cells were incubated in culture medium with 50 nM Mitotracker CMX Red
for 30 minutes at 26°C in the dark. Thereafter, parasites were washed twice with
PBS, resuspended in 1 mL buffer, and analyzed using a cytometer (Beckman
Coulter). Parasites incubated with valinomycin (500 nM for 30 minutes) were used
as positive control 38 (link). To evaluate the
effect of ergosterone coupled molecules on the mitochondrial membrane potential
immediately after molecule addition we used the fluorophore rhodamine-123, a
mitochondrion-specific cationic dye, which distributes across the inner
mitochondrial membranes strictly according to their membrane potential, as
described before 34 (link). Briefly, 1 x
10⁷ parasites were washed twice with rhodamine 123 loading buffer
(20 mM Tris-HCl, 130 mM KCl, 1 mM MgCl₂, and 2 mM
KH₂PO₄) and incubated with 20 μM rhodamine 123 for 45
minutes at 29°C under agitation. Samples were washed twice, resuspended in 500
μL of the same buffer, and transferred to a quartz cuvette. Fluorescence (ex 488
nm and em 530 nm) was registered in a Hitachi F-7000 Spectrofluorimeter. FCCP
was used as control for mitochondrial membrane depolarization 39 (link).

To evaluate mitochondrial membrane depolarization, transduced CRC-SC#1 cells were plated at a concentration of 30,000 cells/well in a 48-well plate pre-coated with Geltrex (Thermo Fisher Scientific, Waltham, MA, USA). Cells were stained with 10 μg/ml JC-1 dye (Adipogen, San Diego, CA, USA) in PBS for 30 min in the dark at 37 °C. FCCP (Cayman Chemicals, Ann Arbor, MI, USA) was added for 15 min at the end of the staining as a positive control. Signals were acquired with a fluorescence microscope (FLoid Cell Imaging Station, Life Technology) and images were analyzed by ImageJ software to determine the red/green fluorescence ratio. Mitochondrial morphology was assessed through Mitotracker CMX-Red (Invitrogen, Waltham, MA, USA). CRC-SC#1 cells were seeded at a concentration of 100,000 cells/well in a 24-well plate pre-coated with Geltrex. Subsequently, cells were stained with 10 nmol/L Mitotracker for 30 min at 37 °C. Fluorescence was visualized by DM5500B fluorescence microscope (Leica, Wetzlar, Germany), and analyzed using ImageJ software v 1.52a (Ashland, OR, USA).

Twenty thousand cells were seeded in 35 mm dish (Microtech) (70% confluence) and leave to adhere overnight. Mitochondrial morphology was assessed after cell staining with 10 nM Mitotracker CMX-Red (Invitrogen) for 30 min at 37°C. Fluorescence was visualized with a digital imaging system using an inverted microscope (Nikon, Japan). Images were captured with a Photometrics Cascade CCD camera system (Crisel) and analyzed with Metamorph acquisition/analysis software. The network morphology was assessed by Metamorph analysis: cells with shape factor ≥0.650 were classified as fragmented network, cells with shape factor <0.650 as tubular network.
Detection of altered mitochondrial membrane potential was performed using 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining kit (Sigma, CS0390) according to manufacturer's instruction.

Mitochondrial morphology was assessed after vital cell staining with 10 nM Mitotracker CMX-Red (Invitrogen) for 30 min at 37 °C. Fluorescence was visualized with a digital imaging system using an inverted epifluorescence microscope with a 63/1.4 oil objective (Nikon, Japan). Images were captured with a back-illuminated Photometrics Cascade CCD camera system (Crisel).