The intracellular Ca2+ level was measured using the Fluo-4-AM (Invitrogen, United States) dye. PC12 cells were stained with 2.5 μM of Fluo-4-AM solution for 30 min in darkness at 37°C and then washed for three times in Hank’s Balanced Salt Solution (HBSS) (Corning, United States). Green fluorescence, which reflects the intracellular Ca2+ level, was recorded with a fluorescence microscope (Carl Zeiss, Germany).
Intracellular ROS levels were detected using fluorescence microscopy with DCFH-DA (Beyotime Biotechnology, Beijing, China). The DCFH-DA was intracellularly deacetylated by a nonspecific esterase, further oxidized by ROS to produce the fluorescent compound 2,7-dichlorofluorescein (DCF) (An et al., 2017 (link)). The analysis was performed with the assay kit protocol. Briefly, PC12 cells were washed twice in DMEM without FBS and incubated with DCFH-DA at 37°C for 25 min. The cells were then washed twice with PBS and analyzed in a fluorescence microscope (Carl Zeiss, Germany).
The intensity of Ca2+ and ROS-labelled cells was further quantified. The labeled cells were counted from five areas under × 20 field or × 40 field randomly chosen from each well using ImageJ software.
Intracellular ROS levels were detected using fluorescence microscopy with DCFH-DA (Beyotime Biotechnology, Beijing, China). The DCFH-DA was intracellularly deacetylated by a nonspecific esterase, further oxidized by ROS to produce the fluorescent compound 2,7-dichlorofluorescein (DCF) (An et al., 2017 (link)). The analysis was performed with the assay kit protocol. Briefly, PC12 cells were washed twice in DMEM without FBS and incubated with DCFH-DA at 37°C for 25 min. The cells were then washed twice with PBS and analyzed in a fluorescence microscope (Carl Zeiss, Germany).
The intensity of Ca2+ and ROS-labelled cells was further quantified. The labeled cells were counted from five areas under × 20 field or × 40 field randomly chosen from each well using ImageJ software.
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