2.1 Cell culture:
BV–2 cell was maintained in Minimum Essential Medium, Alpha 1×with Eagle’s salts, ribonucleosides, deoxyribonucleosides & L-glutamine (MEM; Corning) supplemented with 10% fetal bovine serum (FBS; HyClone) and 1% Sodium Pyruvate(SP 100mmol Gibco NY USA) and 1% penicillin/streptomycin(PS; 100IU/ml penicillin, 100µg/ml streptomycin). N2a cell was maintained in Dulbecco’s Modification of Eagle’s Medium (DMEM; Corning) supplemented with 10% fetal bovine serum (FBS; HyClone) and 1% penicillin/streptomycin (PS; 100IU/ml penicillin, 100µg/ml streptomycin). An incubator with 5% CO2 at 37°C was used to incubate all the cells.
2.2 Lentiviral vector constructs preparation and infection
Lentivirus expressing siRNA(si-Neat1) and control lentivirus(si-con) were packaged by HanBio (Shanghai, China). The packaged recombinant lentivirus then transfected NEAT1 knockdown cells, and then puromycin was used for selection up to at least 15 days.
2.3 OGD/R
Oxygen glucose deprivation (OGD) in microglia was performed as described previously[14]. Briefly, the original culture medium was removed and replaced with glucose/serum-free DMEM at first. Then, the plates were transferred into an anaerobic chamber for 2 h at 37 °C,
which had already been balanced with 5% CO2 and 95% N2 without oxygen. During the reperfusion stage, the plates were then returned to normal medium in normal incubator for 2–72h。
2.4 PCR(Quantitative Real Time PCR)
Total RNA was isolated from the cell using TRIzol (Life Technologies Carlsbad, CA, USA), total RNA of blood was isolated using TRIzol LS Reagent(Life Technologies Carlsbad, CA, USA), cDNA was prepared in 10 µl reaction volume using the ReverTra Ace qPCR RT Kit (TOYOBO CO., OSAKA JAPAN) and real-time PCR analysis run in triplicate using FastStart Universal SYBR Green Master (Rox) (Roche, Mannheim Germany). All the qRT-PCR experiments and data analysis in the present research were performed in accordance with the MIQE guidelines.
2.5 Protein isolation and Western blot assay
Protein samples were isolated from cells and quantified using a bicinchoninic acid (BCA) assay kit (Sigma Aldrich). We used 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the proteins. Proteins were then transferred to a nitrocellulose membrane using an electroblotting technique. The membrane was blocked using a 5% blocking solution (non-fat milk) and then incubated in a blocking buffer with the following primary antibodies overnight at 4°C: Akt, Stat3, P-Akt, P-Stat3, β-actin. The following day, goat secondary antibody conjugated with horseradish peroxidase (HRP) was added to the blocking buffer (1:1,000, non-fat milk), and a chemiluminescence kit (Thermo Fisher Scientific, Shanghai, China) was used to detect the proteins.
2.6 CCK8
N2a cells were plated at a concentration of 20,000 cells/ml in 96-well plates. After exposure experiments, the supernatant of both OGD/R lncRNA Neat1 knockdown BV–2 cells and untreatment lncRNA Neat1 knockdown BV–2 cells were culcured with N2a cells for 24 hr. Then 10 μL CCK–8 reagent (Beyotime, Shanghai, China) were added into each well and incubated for 2 hr at 37°C, a microplate reader (Tecan, SWITZERLAND) was employed to detect the absorbance at 450nm.
2.7 flow cytometry
N2a cells were collected and resuspended in binding buffer. FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, USA) was used and the cells were stained using FITC-Annexin V and propidium iodide (PI). A BD FACSCcantoⅡflow cytometer was carried out to do apoptosis analysis.
2.8 Statistical analysis
All statistical data were analyzed by GraphPad Prism 7.0(GraphPad Software Inc., San Diego, CA, USA). Statistical comparisons were measured by the two-tailed. Student t-test between two groups and by one-way ANOVA with Dunnett’s multiple comparison among more than three groups. Asterisks are used to indicate statistical significance;*, **, ***, and**** indicate p < 0.05, p < 0.01, p <0.001, and p <0.0001, respectively.