The supernatant in each well was removed, and 150 l of DMSO was added and stirred for 10 min

The supernatant in each well was removed, and 150 l of DMSO was added and stirred for 10 min. was from experimental and control group mice via centrifugation at 1200 for 10 min and stored at ?80C. Total cholesterol levels were measured as explained in detail previously 33. Atherosclerotic plaque analysis Mice were killed at 16 and 24 weeks of age, and the aortic root plaques were assessed. After removal of the blood residue, the aortas were quickly dissected and removed from the higher region of the aortic sinus to the lower region of the remaining and right iliac artery. In addition, we retained the heart with the aortic sinus, as well as spleens and cervical lymph nodes (CLNs), which were regularly regarded as the nose\draining lymphatic nodes 33, 38. For plaque analysis, the hearts with the aortic root were inlayed in optimal trimming temperature (OCT) compound for frozen cells sectioning. Six consecutive cryosections (10\m thickness) with three aortic valves were from the aortic root of each mouse and stained with oil reddish O and haematoxylin for lipid visualization. All slices were collected on a Leica CM 1850 Cryostat (Leica Microsystems, Wetzlar, Germany). Plaque area was determined using Image\Pro Plus version 60 software (Press Cybernetics, Rockville, MD, USA). Circulation cytometry analysis At the days 4 and 14 after the final nose administration, splenocytes and CLN cells were harvested from HSP60\treated mice (for 72 h, and the supernatants were collected for detecting the anti\inflammatory cytokines IL\10 and TGF\1; (2) purified CD4+CD25+GARP+ Tregs were co\cultured with CD4+CD25?GARP? T cells (5 103 cells/well) at different ratios (Tregs/Tresps ratios: 1 : 1, 1 : 2, 1 : 4 and 1 : 8) as explained previously 17. The inhibitory effects were measured using an MTT assay. MTT (20 l of 5 mg/ml) was added to each well 4 h prior to harvest. The supernatant in each well was eliminated, and 150 l of DMSO was added and stirred for 10 min. The absorbance (A) ideals were identified at 570 nm on a microplate reader. Dulbecco’s altered Eagle’s medium (DMEM)\F12 culture medium was used as blank control in the normal control group and for zero adjustment of the microplate reader. All measurements were performed in triplicate. The pace of cell proliferation was determined as follows: cell proliferation rate?=?(A value in test group C A value in normal control group)/A value in normal control group 100%. Enzyme\linked immunosorbent assay (ELISA) At the days 4 and 14 and weeks 8 and 16 after the last administration, splenocytes were isolated and cultured with ConA. Untreated or PBS\treated mice were used as settings. The Camptothecin IFN\, IL\4, IL\17, TGF\1 and IL\10 levels in the supernatants were quantified using ELISA packages (eBioscience). In addition, as mentioned above, the levels of IL\10 and TGF\1 in the CD4+CD25+GARP+ Treg tradition supernatant were measured by ELISA. All procedures were conducted following a manufacturer’s instructions. All samples were measured in duplicate. Real\time reverse transcriptionCpolymerase chain reaction Camptothecin (RTCPCR) Total RNA from splenocytes and the descending aortas was prepared using RNAiso Plus (Takara Biotechnology, Shiga, Japan). cDNA was transcribed Camptothecin from purified RNA using a RNA PCR kit (Takara Biotechnology). Actual\time PCR was performed as explained previously 33. The primers are indicated in Table 1. Table 1 Sequences of primers for actual\time reverse transcriptionCpolymerase chain reaction (RTCPCR) 410 687.7??38 DFNA23 7084 m2, respectively; untreated mice; 608??054% 401??038% in spleens, 590??040% in CLNs, untreated Camptothecin mice; 974??092% 401??038% in spleens,.