Male BALB/c mice, 10 weeks old, were purchased from the Animal Center at Shandong University. All experimental procedures were approved by and performed according to the guidelines of the animal ethics committee of the Shandong University School of Medicine. In brief, mice were injected intraperitoneally with streptozotocin (STZ, 60 μg/g) once a day for 5 days to induce diabetes [14, 15]. Control mice were injected with PBS (Phosphate buffer saline). Seven days after the final injection, blood glucose levels were examined by glucometer (UltraVue, Johnson & Johnson, New Brunswick, NJ, USA). Mice with blood glucose levels greater than 300 mg/dl were considered diabetic. Diabetic mice were given LPS (1 mg in 500 μL PBS) orally once to induce inflammation on the 19th day after the first streptozotocin injection, while control mice were treated with the same volume of PBS. 24 hours later mice were killed and intestines dissected and digested with collagenase. Immune cells were isolated by layering on a Percoll gradient, then DCs were further purified using CD11c magnetic beads (130-052-001, Miltenyi Biotec Inc, Bergisch Gladbach, Germany) [16, 17]. Finally the DCs were collected, stained and analyzed by flow cytometry.
Dendritic cells (DC2.4 cell line) were cultured on collagen coated dishes in complete medium containing RPMI-1640, 10% fetal bovine serum, 1 × 104 units/ml penicillin and 10 mg/ml streptomycin. Cells were passaged using standard cell culture techniques. Culture dishes were placed in an incubator equilibrated with 5% CO2 at 37°C. The medium was refreshed at intervals of 3 days. To maintain uniform condition, the cells in passages 20 to 30 were used for experiments.
DC2.4 cells were treated with a high dose of glucose (25 mmol/L) for 48 h. Then LPS (100 μg/ml) was added to detect whether the glucose concentration could affect DC apoptosis. After 24 h LPS treatment (cells grown to approximately 80% confluence), the cells were harvested for both western blot analysis and flow cytometry.
Analysis of apoptosis by flow cytometry
Apoptosis of dendritic cells was determined using an Annexin-V and PI apoptosis kit (BU-ap0102, Life Technologies, Carlsbad, CA, USA). After washing twice with PBS, cells were re-suspended in binding buffer and incubated with Annexin-V and PI for 15 min. At least 5000 cells were counted per sample. Apoptotic cells appeared as Annexin-V positive and PI negative.
Analysis of apoptosis by Hoechst 33258 staining
Hoechst 33258 staining (B33258, Sigma, St Loius, MO, USA) was used to visualize the morphological changes in apoptotic cell nuclei. After being washed with PBS and fixed with 4% paraformaldehyde for 30 min, 0.5 μg/ml Hoechst 33258 was added to the wells and incubated for 5 min. Then cell nuclei were analyzed by fluorescence microscopy. Apoptotic cells were characterized by chromatin condensation and multiple chromatin fragments.
Cells were collected and washed three times with PBS before lysis in lysis buffer (MK163780, ThermoFisher Scientific Inc, Waltham, MA, USA). The protein concentration of the lysates was determined according to the BCA method using the Protein Quantitative Analysis kit (2812 k; CWBIO, CoWin Biotech Co. Ltd., Beijing, China). For electrophoresis, 100 μg of protein was loaded into the wells of a 10% SDS polyacrylamide gel. After separation, proteins were transferred to nitrocellulose membranes, blocked for 3 h at room temperature in blocking buffer (5% nonfat dry milk, Tween-Tris-buffered saline), washed in PBS 3 times and incubated with primary antibody of ABCAM (Rabbit polyclonal to ERK, ab16869, 1: 1:5000; Rabbit polyclonal to AKT, ab66138, 0.15 μg/ml; Rabbit polyclonal to Bax, ab7977, 1:1000; Rabbit polyclonal to Bcl-2, ab18210, 1 μg/ml) and Actin (sc-1616, 1:200, SANTA CRUZ BIOTECHNOLOGY) overnight at 4°C with gentle agitation. Next day after washing three times in PBS, the nitrocellulose membranes were blotted for 1 h at room temperature with secondary antibodies (ab136817, 1:5000, abcam). Finally, immunolabeled protein bands were detected using the ECL(Electro-Chemi-Luminescence) method (Immobilon™ Western, Millipore Corporation, Billerica, MA, USA) and quantified using an Alpha Imager 2200 (ProteinSimple, Santa Clara, CA, USA).
DC2.4 cells were treated as described above. After fixing in 4% paraformaldehyde for 15 min and washing with PBS, cells were permeabilized with 0.1% triton X-100 for 15 min. 3% hydrogen peroxide was used to block endogenous peroxidases. After washing in PBS and blocking in 10% goat serum, cells were incubated with primary antibody of ABCAM (Rabbit polyclonal to ERK, ab16869, 1:1000; Rabbit polyclonal to AKT, ab66138, 1 μg/ml; Rabbit polyclonal to Bax, ab7977, 1 μg/ml; Rabbit polyclonal to Bcl-2, ab18210, 5 μg/ml) and Actin (sc-1616, 1:250, SANTA CRUZ BIOTECHNOLOGY) overnight in a humidified chamber at 4°C. The next day, the cells were washed before being incubated with secondary antibody (SP rabbit HRP kit, CW2035, CWBIO) for 30 min at room temperature. After washing, positive immunostaining was revealed by the DAB(3,3’-diaminobenzidine) method (D8001, Sigma). PBS was used as a negative control for the primary antibodies and Actin was used as a positive control.
Data were analyzed with SigmaStat 3.5 software (SYSTAT Software, San Jose, CA, USA). One-way ANOVA analysis was followed by Dunnett’s test to analyze the differences among the groups. Data are presented as the mean ± SE of at least four independent experiments. P < 0.05 was considered a statistically significant difference.