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… On days 0 and 2, mice were injected i.p. with 200 ng pertussis toxin (cat. no.180; List Biological Laboratories). Mice were evaluated for EAE symptoms every 2 d. The severity of EAE was analyzed according to the following clinical scoring system …
Pertussis toxin (PTX) was obtained from List Biological Labs.
Induction of EAE
MOG35-55-induced EAE in C57BL/6 mice was established as previously described (11–13). In brief, the mice were immunized on day 0 with an intracutaneous injection of 150 mg of MOG35-55 emulsified in CFA-bearing M. tuberculosis strain H37Rv. Injection volume was set at 150 µl. Thereafter, each mouse received an additional 300 ng of PTX by intraperitoneal injection in 100 ml of phosphate-buffered saline (PBS) on day 0 and again on day 2 post-immunization. …
Enzyme-linked immunosorbent assays (ELISAs) were performed to assess antigen and antibody conformations and functional integrity. Briefly, 96-well adhesive plates (Corning) were coated with RTX or pertussis toxin (List Labs) at 0.5 μg/mL in HEPES-buffered saline with 10 mM calcium (HBSC) or 0.2 μg/mL in PBS, respectively, overnight at 4°C. …
HAECs were purchased from Lonza. Cells were grown in EGM BulletKit®−2 (Lonza) at 37ºC in an atmosphere of 5% CO2 and 95% air. The culture medium was composed exactly as recommended by the supplier (Lonza). Confluent cells were exposed to 100 ng/ml LPS (Escherichia coli O111:B4, List Biological Laboratories, #421) for a period within 0.5 h to 24 h as indicated in figure legends. In all experiments, cells of the third passage were used.
In this study, 63 animals were used, divided into 9 groups: control (n = 4), Sham animals (at 1 h (n = 6), 1 day (n = 7), 3 days (n = 7), and 7 days (n = 7) post-surgery), and C2-injured animals (at 1 h (n = 8), 1 day (n = 8), 3 days (n = 8), and 7 days (n = 8) post-injury). None of the C2-injured animals died during or after the procedure, however one Sham animal died at 1 h due to a technical issue. All animals (except n = 4 of each group) received an intrapleural injection with the retrograde tracer Cholera toxin B fragment (CTB, List Biologicals, Campbell, CA, USA) to identify phrenic motor neurons in the cervical spinal cord, similar to that described previously [41,42,43,44]. Briefly, each rat was anaesthetized with isoflurane (Iso-vet, Piramal, Voorschoten, Netherlands; ~1.5% in O2), placed in the supine position on a surgical table while spontaneously breathing into a face mask. The lateral sides of the rib cage were shaved and gently palpated to identify the fifth intercostal space at the anterior axillary line. A sterilized 26G needle connected to a 50 μL Hamilton syringe was used to inject 25 μL of a 0.2% CTB (dissolved in sterile injectable saline) between the 5th and the 6th ribs into the pleural space on each side. …
… Samples were placed in sterile saline (0.9% NaCl) solution with gentamicin sulfate (50 mg/l) and amphotericin B (250 μg/ml). The cartilage samples were transported chilled (approx. 5 °C) to the laboratory. Isolation and expansion of chondrocytes were performed as previously described (Ley et al., 2011). Briefly, the chondrocytes were expanded to passage 1 and then seeded at 20,000 cells/cm2 in chondrogenic medium to maintain the phenotype. On day 4, cells were stimulated with LPS (10 ng/ml, Escherichia coli 055:B5; List Biological Laboratories, Campbell, CA, USA) or kept untreated (controls) for 24 h. Cells were grown to confluence and harvested on day 5 and immediately frozen and stored at −80 °C until further analyses.