Ti s fluorescent microscope

The protein S-glutathionylation induced by 2-AAPA in the TE-13 cells was detected using a detection kit from Cayman Chemical Company according to the manufacture’s instruction. Briefly, TE-13 cells were seeded at densities of 150,000 cells in a 96-well black wall plate. After a 24 h attachment at 37°C, TE-13 cells were treated with growth medium containing various concentrations of 2-AAPA for 1h. Washed the cells twice with PBS and then fixed them with 3.7% formaldehyde in PBS for 10 min at room temperature. Washed the cells once more with PBS and then blocked the protein free-thiol with 100 μL of PSSG Blocking Reagent for 30 min followed by twice wash with 1X Assay Buffer. 100 μL PSSG Reduction Reagent was added to each sample and incubated for 15 min at 37°C. Each sample was washed twice with 1X Assay Buffer and another 100 μL of PSSG Labeling Reagent was added to the samples followed by one-hour incubation. The samples were washed three times with 1X Assay Buffer and incubated with PSSG Detection Reagent II (FITC) at a 1:50 dilution in 1X Assay Buffer for 1 hour. The cells were washed twice with 1X Assay Buffer and the fluorescent images were captured using a Nikon Ti-S fluorescent microscope.

Remaining testes from the same litter were decapsulated and single cell suspensions were obtained following a 2-step enzymatic digestion procedure as previously described (Rwigemera et al., 2017 (link)). A trypan blue-stained subset of this single cell suspension was used to determine cell concentration and viability. Cells were then diluted to reach an optimal concentration of 5-10 million cells/ml for cell sorting using a FACSJazz flow cytometer (BD Biosciences, San Jose, CA) at an event rate of ∼2,500 events/sec as described in (Rwigemera et al., 2017 (link)). After sorting, the GFP-positive cell fraction was washed with Hanks’ Buffered Salt Solution and counted. The total number of cells collected, viability and purity per fraction (percentage of GFP-positive cells) were calculated using a hemocytometer under a TiS fluorescent microscope (Nikon, Mississauga, Ontario, Canada). Sorted cells were finally aliquoted, pelleted, snap frozen in liquid nitrogen and stored at −80°C until RNA extraction.

A549 cells were plated in 48-well plates at a density of 1.5 × 10⁴ cells per well and grown overnight. The dilutions of test compounds prepared in DMSO and dissolved in the culture medium were added to the cells in triplicate for each concentration so that every well contained 200 μL of conditioned medium and 200 μL of fresh medium with the test substance and incubated for 2 h. The final DMSO concentration was 0.5%. After the incubation, the cells were stained using a Apoptosis/Necrosis assay kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s protocol and imaged using a Nikon Ti-S fluorescent microscope.