Il 8 antibody

PIEC migration was determined as previously described [29 (link)] with minor modifications. PIECs were seeded (4×10⁵cells/well) into 6-well plates and cultured for 24 h under static conditions. The confluent EC monolayer was scraped in a straight line with a 200 μL pipette tip to create a wound parallel to the direction of the flow. Wounded monolayers were washed once with 1 mL of growth medium to remove cell debris. The in vitro scratch wound healing assay was performed under four different conditions including a control group, PCV2-infected group, IL-8-antibody group, and PCV2-infected-IL-8-antibody group (n = 3 wells per group). PCV2-infected groups were infected as described above. IL-8 antibody was added into the cell supernatants at a final concentration of 3 µg/mL (Abcam, Cambridge, UK) to the relevant groups. The cells were grown in a humidified 5% CO₂ atmosphere at 37°C for 24 h. Images of the cell monolayer around the wounds were taken with an Olympus inverted phase microscope at 24 hpi (Olympus, Tokyo, Japan).
Data were presented as wound closure (%): Wound closure [(A0 -At)/A₀]×100% where A₀ is the area of the original wound and A_t is the area of wound measured 24 hpi. All experiments were repeated at least three times.

To screen the optimal concentration of inhibitors, the induced DCs were cultured in complete medium with 2 μM, 5 μM, 10 μM, or 20 μM of BAY 11-7082 (Beyotime, Shanghai, China) or Ruxolitinib (Selleck Chemicals, Houston, TX, USA). After 1 h of incubation, the culture medium was discarded, washed three times, and then 10% RPMI1640 induction medium was added to the upper chamber of the transwell membranes for a further 48 h. These MoDCs were collected to detect IL-12 mRNA and determine their optimal concentration. DCs treated at the optimal concentration were co-cultured with PIECs of different treatments. To minimize the impact of other factors, we established an IL-8 antibody panel by adding IL-8 antibody (Abcam, Cambridge, MA, USA) at a final concentration of 5 μg/mL to the co-culture. The six groups in the NF-κB signaling pathway experiment included PCV2-PIECs-DCs, IL-8Ab-PCV2-PIECs-DCs, IL-8Ab-PCV2-PIECs-BAY-DCs, IL-8^over-PIECs-DCs, Ab-IL-8^over-PIECs-DCs, and Ab-IL-8^over-PIECs-BAY-DCs. The six groups in the JAK-STAT signaling pathway experiment included PCV2-PIECs-DCs, IL-8Ab-PCV2-PIECs-DCs, IL-8Ab-PCV2-PIECs-RUX-DCs, IL-8^over-PIECs-DCs, Ab-IL-8^over-PIECs-DCs, and Ab-IL-8^over-PIECs-RUX-DCs. The DCs of different groups were used for flow cytometry and quantitative real-time PCR.

HAECs that were either treated with Mox-LDL (100 µg/mL) or left untreated, were seeded onto coverslips and fixed with 4% PFA at 4 ºC. Cells were washed with PBS (1X) 3 times. Then, they were blocked for 1 h with PBS (1X) and 3% normal goat serum (NGS) and incubated again for 3 h with interleukin-8 (IL-8) antibody (abcam, USA) prepared in PBS (1X) and 1% NGS. Cells were further incubated with Alexa fluor 488 secondary antibodies (Invitrogen, USA) prepared in PBS (1X) and 1% NGS for 1 h. Nuclei were counterstained with Hoechst 33,324 (Thermo Fisher, Molecular Probes, Eugene, OR, USA). Coverslips were mounted onto glass slides using Prolong Antifade (Molecular Probes) and slides were observed under the LSM 710 microscope at 40× magnification.