Z1 inverted microscope

The effect of chemicals on cell GSH content was determined by fluorescence microscopy. Briefly, 0.5 × 10⁶ cells/mL were plated in a 12-well plate, exposed to chemicals during indicated times, washed with PBS and then loaded with 30 μM dibromobimane for 30 min at 37 °C in the dark. Cells were then washed with PBS and transferred to µ-slides 8-well ibidiTreat (ibidi GmbH, München, Germany) for observation. Images were obtained using an Axio Observer. Z1 inverted microscope (Zeiss) and analyzed with Fiji software from ImageJ (http://fiji.sc/Fiji).

HTR-8/SVneo cells were incubated in a 6-well plate at a density of 5 × 10⁵ cells per well. When the cell confluence reached 80 %, the cells were scratched with a 200 μl pipette tip, washed three times with PBS, and then RPMI 1640 medium containing 2 % FBS was added. The scratch sizes of cells at 0 h, 12 h, and 24 h were recorded with a Z1 inverted microscope (Carl Zeiss, USA). The percentage of wound healing was analyzed using ImageJ software (version 1.53; National Institutes of Health, USA). All experiments were repeated independently three times.

The level of mitochondrial-derived superoxide radicals in the mPMs exposed to HIV TAT was monitored using the MitoSOX™ Red dye (Invitrogen; M36008) according to the manufacturer's instructions. Briefly, MitoSOX™ Red reagent permeates live cells where it selectively targets mitochondria and is rapidly oxidized by superoxide, but not by other ROS or reactive nitrogen species. Oxidation of MitoSOX™ Red reagent by superoxide produces red fluorescence that is visualized by microscopy. Herein, mPMs seeded on sterile glass coverslips (11 mm) placed in a 24-well plate at a density of 5 × 10⁴ cells per well and exposed to HIV TAT (7 nM) for 48 h. Followed by the cells were washed with 1x PBS and incubated with MitoSOX™ Red dye (5 μM) for 15 min at 37 °C. After staining, the cells were washed and fluorescent images were acquired on a Zeiss Observer using a Z1 inverted microscope (Carl Zeiss, Thornwood, NY, USA). As a precaution, images were acquired within 10–20 min, since MitoSOX tends to accumulate in the nucleus after approximately 40 min [57 (link)]. All the experiments were repeated six times.