2.1. Human samples
40 human serum samples (20 PD patients and 20 healthy people in the same age range) were obtained from Jiangsu Taizhou People’s Hospital. The collection and use of all clinical samples were approved by the Clinical Research Ethics Committee of Taizhou People’s Hospital. All patients and healthy people had no history of other inflammatory diseases and anti-inflammatory drugs within the past 2 months. Serum HMGB1 and CXCL12 were detected according to the protocol of the ELISA kits (Solarbio; MultiSciences). For the correlation analysis of clinical data, we used Pearson’s chi squared (χ2) Test. p < 0.05 was considered statistically significant. For human serum co-immunoprecipitation, serum sample was first placed in 100kDa Amicon Ultra-15 (Millipore) and centrifuged at 1000rcf for 20 minutes to obtain IgG-depleted PD patient serum. Subsequently, the samples were incubated with anti-human HMGB1 monoclonal antibody (ab228624, Abcam) and Protein A+G agarose beads (P2012, Beyotime); after thorough washing, protein loading buffer was added to the agarose beads for a boiling water bath for 5 min, and then SDS-PAGE electrophoresis was performed, and CXCL12 was detected by antibody.
For the isolation of CD3+ CD14+ cells from human peripheral blood, first centrifuge the peripheral blood at 500 rcf for 10 min, lyse the red blood cells with ACK, then resuspend the cells in buffer, incubate overnight in CXCR4 antibody or IgG from the same species, and add Protein A+G agar after centrifugation and washing. The sugar beads were incubated for 3 hours, and then centrifuged and washed several times. The agarose beads were reselected with protein loading buffer, and the beads were bathed in boiling water for 5 minutes, and then HMGB1 and CXCL12 was detected by SDS-PAGE electrophoresis.