N13 microglia cell line was prepared in the frozen liquid nitrogen and then put into the water with a temperature of 37℃ for shaking 30 seconds (s). The whole cell suspension was placed into the centrifuge tube that contained 30 ml Dulbecco's modified Eagle's medium (DMEM), penicillin (100 IU/mL), streptomycin (100 IU/mL) and 10% fetal bovine serum (FBS). After centrifugation at 1000 rpm for 5 minutes (min) at room temperature (RT), microglia suspension was plated in one T75-cell culture flask (Invitrogen, Carlsbad, CA) in the DMEM medium and incubated at 37℃ and 5% carbon dioxide (CO2) in a humidified incubator. The medium was changed every 2 days until the cells became confluent. When the cell layer reached full confluence, microglia were harvested using pre-warmed trypsin (Sigma) to prepare for the experiment.
For stimulation experiments, microglia were harvested from the remaining cells, seeded in 6 well plates in DMEM with 10% FBS, and set up three different time point groups for 24, 48, 72 h, and the cell count were 2×105, 105, 5×104 cells/well respectively and then stimulated for 12 h. Microglia were divided into treatment groups: (a) unstimulated state (resting microglia phenotype); (b) polarized to pro-inflammatory phenotype induced by 500 ng/ml lipopolysaccharide (LPS; Sigma) and 20 ng/ml recombinant mouse interferon (IFN)-γ (ImmunoTools GmbH, Friesoythe, Germany); (c) polarized to anti-inflammatory phenotype produced by stimulation with 20 ng/ml recombinant mouse IL-4 and IL-10, as well as recombinant human TGF-β (R&D systems, USA) for 24 h. And further stimulated with or without 200 μM KA for 24, 48, 72 h. After stimulating for 24, 48, 72 h, the supernatants were aspirated for LDH activity test, as well as the detection of NO and cytokine levels, and the microglia were analyzed by flow cytometric to check the polarization of resting microglia to pro-inflammatory or anti-inflammatory phenotypes according to standard protocols.
Detection of polarization of resting microglia to pro-inflammatory or anti-inflammatory phenotypes analyzed with Fluorescence-activated cell sorting (FACS)
The microglia were washed with phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA; Sigma) twice and stained with combinations of cell surface antibodies for 30 min at 4 °C. The used antibodies included: APC-labeled rat anti-mouse CD11b (Caltag, Burlingame, CA), rat anti-mouse Arg-1 (BD Transduction, USA), FITC-labeled rat anti-mouse CD206 (Biolegend, USA) and PE-labeled rat anti-mouse CD40 (Caltag, Burlingame, CA), FITC-labeled rat anti-mouse inducible NO synthase (iNOS) (BD Transduction). For intracellular staining, 5000 cells/tube with good conditions were first incubated with the surface antibodies, fixed with 2% buffered formaldehyde for 20 min at 4 °C, and permeabilized with 0.5% saponin in PBS/BSA. FITC-, PE-, APC-labeled mouse isotype IgG (Serotec) were used as negative controls. The percentages of CD11b, Arg-1, CD206, CD40 and iNOS positive microglia were detected using BC CytoflexS flow cytometer and the data were analyzed using CytExpert 2.2 software (Beckman Coulter, USA).
Detection of cell activation with Cell Counting Kit-8 (CCK-8) Test
The culture supernatants were transferred into a new 6-well culture plate. Fresh medium with 10% CCK8 was added and incubated for 1-4 hours at 37℃. The absorbance was measured at 570 nm with a reference filter of 570 nm.
Detection of LDH Activity with LDH Kit
The total protein concentrations were measured by using the bicinchoninic acid (BCA) protein assay kit (Bio-Rad, Sweden). The standard curve was obtained by using BSA solutions at concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 mg/ml respectively. The concentrations of proteins were quantified from the standard curve.
LDH production was detected in the culture supernatants by using Cytotoxicity Detection Kit and following the kit directions. Briefly, 100 μl assay medium was added into triplicate wells for background control, while those wells with the same amount of supernatant from untreated common cells or Triton X-100 treated cells included were for low control or high control. 100 μl samples were added to each well. 100 μl of the freshly prepared reaction mixture was added into each well and incubated for 30 min at RT away from light. The absorbance of the samples was measured at 490 or 492 nm.
Detection of NO Production
NO production was measured by the supernatant levels of nitrite, while the stable biological oxidation product of NO was determined by using the modified Griess reagent. The concentrations of nitrite were quantified by the extrapolation from the standard curve, which was obtained by using sodium nitrite solutions at concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0 μM. The samples and Griess reagents were added each 100 μl to each well. After being incubated 15 min at RT away from light, the absorbance of the samples was measured at 540 nm.
Detection of cytokine levels with Enzyme-Linked Immunosorbent Assay (ELISA)
The cytokines levels (TNF-α, IFN-γ, IL-4, IL-10, and IL-6) in the supernatant of microglial cultures before and after KA insult were assessed using commercially available kits (eBioscience) on an ELISA reader at 450 nm following the standard manufacturer's protocols. 100 µl/well of capture antibody (2 µg/ml TNF-α, 1 µg/ml IFN-γ, 2 µg/ml IL-4, 1 µg/ml IL-6, and 2 µg/ml IL-10) was used to coat the plates at 4°C overnight. After being washed with Wash Buffer, uncoated sites were blocked by 200 µl/well of 1X ELISA Diluent for 1 h at RT. After being incubated at room temperature for 1 h, wells were aspirated and then washed 3 times with Wash Buffer of at least 300 µl/well. Duplicates of samples or of recombinant standards were added and the plates were incubated overnight at 4°C. The plates were incubated with detection antibody (1 µg/ml TNF-α, 1 µg/ml IFN-γ, 1 µg/ml IL-4, 1 µg/ml IL-6, and 1 µg/ml IL-10) at RT for 1 h after being washed. Then avidin-conjugated horseradish peroxidase (HRP) was added for 30 min at RT. 100 µl of 3,3',5,5'-Tetramethylbenzidine (TMB) was added to each well and incubated at RT for 10-15 min. The color reaction was stopped by the addition of 100 µl of 2 M sulfuric acid and read at 450 nm.
Data Presentation and Statistics
Each data was presented as mean value ± standard deviation (SD). The statistical program for social sciences 15.0 (SPSS 15.0, USA) was used to analyze the experimental results. Analysis of Variance was used to compare values among groups. For all tests, the level of significance was set to P< 0.05 which is shown on the graphs as *P< 0.05, **P< 0.01 and ***P < 0.001. Statistics used for each data are indicated in the figure legends.