Citations

Enzyme-linked immunosorbent assays

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. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

… On the first and third days, the mice received 100ng of pertussis toxin (List Biological Laboratories) …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

2.1. Cell culture and treatment

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.

• Product #421 – ULTRA PURE LPS from Escherichia coli O111:B4

2.2. Animal Groups and Surgical Preparation

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. …

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt

Reagents

The following reagents were obtained as indicated: … lipopolysaccharide (List Biological Laboratories, Escherichia coli O111:B4), …

• Product #201 – LPS from Escherichia coli O111:B4

2.1. Chondrocyte isolation and culture

… 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.

• Product #203A – LPS from Escherichia coli O55:B5

… goat anti-CTB (1:2k, List Biological Laboratories, catalog # 703, RRID: AB_10013220), …

• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

4.6. Serological analysis of B. pertussis specific antibodies

On the day of processing, a cardiac puncture was performed on each mouse. Approximately 1 mL of blood was removed from each mouse. The samples were centrifuged for 2 min at 14,000 × g. Serum was collected and stored at − 80 °C until analyses were performed. Serological responses specific to B. pertussis antigens were quantified by ELISA. High-binding microtiter plates were coated with PT (50 ng/well) (PT#180, LIST Biologicals), FHA (50 ng/well) (Enzo Life Sciences), or PRN (50 ng/well) (PRN#187, LIST Biologicals) as described in Boehm et al. [52]. …

• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #187 – Pertactin from B. pertussis (69 kDa Protein)

… There followed an incubation in anti-CT-B made in goat (List Biological #703; 1:4000 dilution in TBS-TX) and 3% horse serum for 2-3 days at 4°C …

• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

… After EAE induction, intraperitoneal injections of 200 ng pertussis toxin (List Biological Laboratories, USA) in the first hour and 400 ng pertussis toxin in 200 µl PBS were administered at the 48th hour. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)