Bacterial Toxin Research Citations

Induction of EAE model

Female Lewis rats at 6-8 weeks of age were purchased from Beijing Vital River Laboratory Animal
Technology Co., Ltd (Beijing, China). The antigen emulsion was prepared from an emulsion of guinea pig
spinal cord white matter homogenates in phosphate-buffered saline (PBS, 50% w/v) mixed with an equal
volume of complete Freund’s adjuvant (Sigma-Aldrich) supplemented with 4 mg/mL Mycobacterium
tuberculosis. Each rat was immunized by subcutaneous injection of antigen emulsion at a dose of 0.75
ml/kg into its footpad, followed by an auxiliary injection of 8 μg/kg diluted in 0.5 mL pertussis toxin (List
Biological Laboratories) in the dorsum of the foot. The second immunization was administered 7 days
after the first immunization. …

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)

EAE

Pertussis injection solution (per mouse)

Reagent Stock concentration Final concentration Amount
Pertussis 100 μg/mL 2 μg/mL 2 μL

Pertussis toxin List Biological Laboratories Cat# 181

• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)

… 0.1mL PBS containing 200ng pertussis toxin (List Biological Laboratories) was administered intravenously at day 0 and day 2 post immunization per mice. …

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)

… complete Freund’s adjuvant containing 4 mg/mL H37Ra M. tuberculosis was used for C57BL/6J and CD38KO mice, followed by the administration of pertussis toxin (200 ng per mouse; List Biological Laboratories, Inc.) …

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)

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