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.