Fluo 4 acetoxymethyl ester fluo 4 am

All odorants used in the present study were purchased from (Sigma-Aldrich, St. Louis, MO, USA) and were of the purest grade (>95% pure). Stock solutions (100 mM) of the odorants were prepared using dimethyl sulfoxide (DMSO) and stored at −20 °C. For each assay, odorant solutions were freshly diluted from the stock solution to the desired concentration in DMSO. Fluo-4-(acetoxymethyl) ester (Fluo-4 AM) (excitation at 494 nm, emission at 516 nm), obtained from (Beyotime, Shanghai, China) as a lyophilized powder, was diluted to 1 mM using DMSO and stored at −20 °C. The composition of the calcium assay buffer was as follows: 21 mM KCl, 12 mM NaCl, 18 mM MgCl₂, 3 mM CaCl₂, 170 mM D-glucose, 1 mM probenecid (Sigma-Aldrich, St. Louis, MO, USA), and 10 mM piperazine-1,4-bisethanesulfonic acid. The pH of the buffer was adjusted to 7.2, and the buffer was filter-sterilized (using a 0.22 µm filter) prior to use.

Fluo-4 acetoxymethyl ester (Fluo-4-AM) (Beyotime, Beijing, China) dye was used to measure intracellular calcium. HT22 cells were seeded into 6-well plates at a density of 2× 10⁵ cells/well and treated as described above. After treatment, the cells were stained with Fluo-4-AM diluted to a concentration of 2.5 μM for 30 min in darkness at 37 °C and then washed with Dulbecco’s phosphate-buffered saline (DPBS) three times. The green fluorescence, which reflected the intracellular calcium level, was recorded by a fluorescence microscope (Olympus, Japan).
An MMP assay kit with JC-1 (Beyotime, Beijing, China) was used. The kit resulted in the formation of JC-1 aggregates that emitted red fluorescence at a high MMP, while monomers that emitted green fluorescence at low MMP were produced when they selectively entered the mitochondria. After treatment as described, the cells in different groups were washed with PBS twice and then incubated with 2 μM JC-1 in fresh culture medium under permissive conditions. After 30 min, the cells were washed with PBS three times and resuspended in culture medium. Red and green fluorescence was detected by a fluorescence microscope (Olympus, Japan).