Ti e confocal microscope

MEFs were seeded in SensoPlate™ 24-Well Glass-Bottom Plate at the density of 50,000 cells per well and cultured for 24 h. Then cells were treated with 1X MagicRedTM (ImmunoChemistry Technologies, #937) staining solution for 30 min at 37 °C. Cells were analyzed with a Nikon Ti-E confocal microscope equipped with A1R scan head with spectral detector and resonant scanners using TRITC filter. Images were quantified using Image J.

MEFs were seeded in SensoPlate™ 24-Well Glass-Bottom Plate at the density of 50,000 cells per well and cultured for 4 h. The cells were treated with different concentrations of acetyl-l-carnitine for 20 h. MitoTracker® Green FM (Invitrogen, M7514) was added to cultural medium to make a final concentration of 50 nM MitoTracker® Green FM. Cells were incubated with MitoTracker® Green FM at 37 °C for 30 min; then the medium was removed and washed twice with PBS. Cells were analyzed with a Nikon Ti-E confocal microscope equipped with A1R scan head with spectral detector and resonant scanners using FITC filter. Images were quantified using Image J.

The cells were seeded into 15 mm confocal dishes, and immunofluorescence staining was performed after the intervention. MitoTracker Red CMXRos (Life Technologies, Carlsbad, CA, USA), diluted in serum-free medium to 100 nM, was added to the cells, which were then incubated in the dark at 37 °C for 30 min. ER Tracker Blue White DPX dye (Life Technologies, Carlsbad, CA, USA), diluted in serum-free medium to a final concentration of 200 nM, was then added to the cells, which were incubated in the dark at 37 °C for 30 min. Next, the cells were fixed with 4% paraformaldehyde for 15 min and permeabilized using 0.3% after washing three times with PBS. Triton for 15 min, and sealed with 5% BSA for 30 min. Primary antibodies (1:100) were added. The cells were incubated in the dark at 4 °C overnight. DyLight 488 goat anti-rabbit IgG (1:200; A23220; Abbkine, Wuhan, China) was then added, and the cells were incubated in the dark at 37 °C for 1 h. Images were captured using a TiE confocal microscope (Nikon, Tokyo, Japan). A plot profile was generated using ImageJ software to assess the degree of colocalization.

P. falciparum was cultured at 37 °C under 5% CO₂, 5% O₂ and 90% N₂ in the presence of 20 μM ZnMP for 1 h, and then red blood cells were washed by PBS to remove ZnMP in the medium. ZnMP uptake was examined under a Nikon Ti-E confocal microscope equipped with a ×100 MPLAPON oil objective with the excitation wavelength at 488 nm and the emission spectrum at 550–650 nm.