1. Cell culture and treatments
The1321N1 human astrocyte cell line (American Type Culture Collection, USA)wascultured in DMEM-F12 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Beyotime Institute of Biotechnology, Shanghai, China) at37 °Cin 5% CO2. After 2 daysofincubation,thecells were incubated with serum-free DMEM-F12 medium for 24hours and thenchallenged with20 μMTNFα (Sigma–Aldrich, St. Louis, MO, USA) for 24 hours after being preincubated with phosphate-buffered saline (PBS, naïve control),1nMDex (Sigma–Aldrich, St. Louis, MO, USA) or10 nMAtipamezole (α2 receptor antagonist) (Ati, MedChem Express, Monmouth Junction, NJ,USA) mixedwith1 nMDex for 15 minutes. TNFα and Dex concentrations were chosen based on our previous study5, and the dose of Ati was based on an agonist-antagonist binding affinity ratioof10:1.
2. Cell cycle analysis by flow cytometry
The cell cyclewasanalyzed by flow cytometry (ACEA Biosciences Inc, San Diego, California, USA)9. In brief, the cells were detached fromtheflask after trypsinization and then fixed with 70% ethanol at4 °Cfor 12 hours.After being centrifuged at2500 rpm,thecells were suspendedin0.5 ml of PBS containing 500 ng/L RNase and 10𝜇L of40𝜇g/L propidium iodide(PI) and incubated for 10 minutes inadark box at room temperature. Fluorescence was measured by flow cytometry in at least 10,000 gated events andwasanalyzed by FlowJo 7.6.1 software.
Cells were incubated on cover slips and washed with 0.01% PBS3 timesfor 5minutes each,followed bytreatmentas previously described. Then, thecells were fixed with 4% paraformaldehyde (PFA) for 10 minutes and washed again. After being blocked with donkey serum for 10 minutes,thecells were incubated with monoclonalantibodies,including GFAP and α2antibody mixtures(for double labeling), cyclins A, B, D and E, Ki67,andcleaved caspase 8 and 9(1:200, Abcam, Cambridge, MA, USA),overnight at4 °C. Afterthe primaryantibody was removed and the cells were washed again,afluorochrome-conjugated secondary antibody (Abcam, Cambridge, MA, USA) was added and incubated for 1 hour. Then, the cellnuclei werecounterstained with DAPI,andthe slides were examined using an Olympus IX71 microscope underaconstant exposure level. Each experimentwasrepeated 4 times,and 10 areas were chosen randomly on each slide. The number of positive cells relative to the number of DAPI-positive cells and immunofluorescence levels were quantified using ImageJ (National Institutes of Health, Bethesda, MD).
4. Detection of △ψm
Astrocytes were labeled withthelipophilic cationic probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanineiodide (JC-1) and analyzed byfluorescence microscopyor flow cytometry previously described2.To investigatethe direct visual changesinfluorescence after JC-1 staining, cells were incubated on cover slips, treated asadivided group and visualized underamicroscope. In brief,the coverslipswerecarefullywashed with warm PBS twice and then incubated with 0.2 μM JC-1 for 30 minutes at 37 °C inadark box. Subsequently, thecoverslipswere washed again and moved to glass slides,and then immunofluorescence imageswereobtained withanOlympus IX71 microscope. Red and green fluorescence intensities inthesame visual areawereanalyzed with ImageJ software,andthemean fluorescence intensity (MFI) was calculated from 4 areas chosen randomly in each slide.Tofurtherinvestigatethe proportion of cells with high△ψm, cells in various groups were collected by trypsinization and then transferred to 5-mL polystyrene tubes for flow cytometry. After being washed with fluorescence-activated cell sorting (FACS) buffer (10% fetal calf serum, 0.5 M EDTA in 0.1 M PBS) once, the cells were incubated with 0.2 μM JC-1 in FACS buffer for 30 minutes at 37 °C inadark box. The cells were analyzed by flow cytometry after being washedtwicewith warm FACS buffer. Fluorescence was measured in the FL-1 (green fluorescence) and FL-2 (red fluorescence) channels after gating on live cells. The mean intensities of red fluorescence (PE) and green fluorescence (FITC) were analyzed using FlowJo 7.6.1 software,and the ratio of red/green fluorescence intensity was analyzed using GraphPad Prism 8 (GraphPad Software, San Diego, CA). The cells withahigh red/green ratio were gated relative totheNC group.
5. Flow cytometric analysis of ROS generation
The changesinROS production in naïve control and treated cellswere assessedusing flow cytometry after 2’-7’-dichlorodihydrofluoresceindiacetate (DCF, Invitrogen, Paisley, UK) staining. In brief, astrocytes were harvested by trypsinization and then incubated in 2𝜇M DCF diluted in FACS buffer for 30 minutes at37 °C. The fluorescence intensity was assessed by flow cytometry and analyzed with FlowJo 7.6.1 software. Each assay included at least 10,000 gated events.
Allexperiments wererepeated at least 4 times independently.
All numerical data are presented as the mean ± SD or box-whisker plots. Comparisons between the treatment groups were analyzed by one-way analysis of variance followed by Tukey’s multiple group comparisons (equal variances) or the Kruskal–Wallis (nonparametric) test followed by Dunn multiple group comparisons (unequal variances or scoring) (GraphPad Prism 5, San Diego, CA). A p value of 0.05 was considered statistically significant.