Mitotracker

A 6-well plate had its bottom sawed off and hand-filed down. A #1 microscope coverslip (Ted-Pella, Redding, CA) sufficient to cover the 35 mm² area was adhered with aquarium seal and allowed to dry over-night. The plates were re-sterilized with ethanol and UV for 48 hr prior to use. 1 hr before cell plating, 0.1% gelatin in PBS was pipetted onto the cover glass and incubated at 37°C. HAECs were then plated onto the coverglass overnight before flow experiments. After flow experiments, the media was changed to EGM-2 with either 5 mM CellRox Orange (ThermoFisher) or 500 ${\displaystyle \mu }$ M MitoTracker Deep Red (ThermoFisher) and placed in the incubator for 30 min. The media was changed again for EGM-2 and the cells imaged immediately. A standard mercury lamp and TRITC (CellRox) or Cy5 (MitoTracker) filter set was used on an inverted microscope (Zeiss Axio 200).

Fluorescent probes that stained different cellular compartments were used to characterize the origin of the vacuoles. The dapoxyl ER-tracker blue-white dye, the Lyso-tracker red dye, the Mito-tracker green dye, and 10 kDa and 70 kDa Texas-Red labeled dextran were all obtained from Molecular Probes (Life Technologies, Merelbeke, Belgium). We also used Lucifer yellow (Lucifer Yellow CH, lithium salt) from Biotium (Fremont, CA, USA) and acridine orange from Sigma-Aldrich. Briefly, the cell seeding and treatment procedures were similar to the ones used for the phase contrast microscopy (Section 4.3.1.). The dye solutions were simply added to the culture medium 1 h before the end of the treatment periods, excepted for the Lucifer yellow and both dextrans 10 kDa and 70 kDa, which were added for the whole duration of the treatment. The concentrations of the dyes were as follows: ER tracker, 0.5 µM; Lyso tracker, 75 nM; Mito tracker, 200 µM; Lucifer yellow, 100 µg/mL; acridine orange, 1 µg/mL; and dextrans 10 kDa and 70 kDa, 125 µg/mL; At the end of the treatment period, the procedure was similar to that of phase-contrast microscopy to take pictures of living cells with the Imager M2 fluorescence microscope (Carl Zeiss) coupled with the AxioCam ICm1 and AxioImager software (Carl Zeiss). The experiment was realized at least two times in duplicate.

Exponentially growing parasites in regular growth medium were collected and resuspended in 10 mL PBS-G (1X PBS, 0.1% glucose). Cells were transferred to flasks and MitoTracker Deep Far Red (ThermoFisher M22426) added at the final concentration of 250 nM. Cells were incubated for 20 minutes at 27°C, collected and fixed with paraformaldehyde and counter-stained with DAPI, and then slides were prepared as described previously [12 (link)]. Slides were visualized on a Carl Zeiss Laser Scanning Microscope 710 using 63x oil DIC objective and MitoTracker excited with the 637nm laser. DAPI was excited with the 405-diode laser. Attained images were analyzed using ZEN 2.3 lite software. Cells were qualitatively compared for the differences in mitochondrion morphology. Representative images from 3 biological replicates are presented.