Emical analyzer (Olympus, Tokyo, Japan). Serum TNF-, IL-1 and IL-6 levels
Emical analyzer (Olympus, Tokyo, Japan). Serum TNF-, IL-1 and IL-6 levels were determined employing enzyme-linked immunosorbent assay (ELISA) kits (Boster Biological Technology Ltd, Wuhan, China) based on the manufacturer’s protocol. Cell Death and DiseasePer1 alleviates excessive hepatic immune response T Wang et alAntibody. For immunohistochemical staining, purified antibodies were obtained from Abcam (Shanghai, China) and eBioscience (San Diego, CA, USA) (anti-mouse CD68, Abcam, cat no. ab955; anti-mouse F4/80, eBioscience, cat no. 14sirtuininhibitor801). Antibodies for flow cytometry analysis were obtained from eBioscience and BD Biosciences (San Jose, CA, USA) (anti-mouse F4/80, PE-conjugated, eBioscience, cat no. 12sirtuininhibitor801; anti-mouse CD11b, FITC- conjugated, BD Biosciences, cat no. 557396; anti-mouse CD115, APC-conjugated, eBioscience, cat no. 17-1152). Antibodies for western blots and immunoprecipitation assays were obtained from Sigma-Aldrich, Cell Signaling Technology (Shanghai, China), Abcam and Bioworld Technologies (Nanjing, China) (anti-HA, Sigma-Aldrich, cat no. H3663; anti-mouse PPAR, Cell Signaling Technology, cat no. 2430; anti-mouse PER1, Abcam, cat no. ab3443; anti-mouse -actin, Bioworld Technology, cat no. AP0060). Histological evaluation. Liver tissue was fixed in ten phosphate-buffered formalin overnight, embedded in paraffin and cut into 4-m sections. The sections had been stained with hematoxylin and eosin (H E). CD68 and F4/80 have been used as immunohistochemical markers for KCs inside the liver as described previously.46 Cell culture and treatment. Peritoneal macrophages have been isolated from mice by peritoneal lavage four days right after injection of 2 ml of 3 thioglycolate as described previously.47 Peritoneal macrophages and RAW264.7 cells were maintained in RPMI-1640 supplemented with 10 low-endotoxin FBS and stimulated with LPS (1 g/ml) for the indicated time periods. RNA extraction and quantitative real-time PCR. Total RNA was Clusterin/APOJ Protein Molecular Weight extracted from the samples with Trizol (Invitrogen, Carlsbad, CA, USA) in line with the manufacturer’s instructions. The reverse transcription reaction was carried out employing a reverse transcriptase kit (Invitrogen) in line with the manufacturer’s protocol. Real-time PCR was performed, along with the PTPRC/CD45RA, Human (HEK293, His) solutions have been detected utilizing the ABI 7300 Detection Program with SYBR Green dye (Toyobo, Osaka, Japan). The expression amount of glyceraldehyde-3-phosphate dehydrogenase was simultaneously quantified as an internal typical control. Gene expression in monocytes/ macrophages was normalized against B2M and TBP as previously described.48 The sequences of all primers made use of for quantitative RT-PCR are shown in Supplementary Table S1. Flow cytometry. For flow cytometry experiments, hepatic inflammatory cells and peripheral blood mononuclear cells (PBMCs) had been ready as described previously.29 Hepatic inflammatory cells were stained with fluorescently labeled antibodies against F4/80 and CD11b. PBMCs had been incubated with fluorescently labeled antibodies against CD115 and CD11b. Flow cytometric evaluation was performed by utilizing a FACScan flow cytometer (BD Biosciences) at Nanjing Healthcare University. For cell cycle analysis, cells within the G0/G1, S and G2/M phases in the cell cycle had been identified employing Vybrant Dye Cycle violet stain (Invitrogen) based on the manufacturer’s protocol. The proportion of apoptotic cells was measured using a FACScan flow cytometer based on the guidelines provided in the Annexin V/P.