Citations

Lactate dehydrogenase (LDH) assay:

Macrophages were pretreated for 30min with glycine or one of the other small-molecule inhibitors at the indicated dose. All chemicals were purchased from Sigma-Aldrich, with the exception of propofol (EMD Millipore). Late-log cultures of Salmonella typhimurium SL1344 grown in L-broth containing 0.3M NaCl were used to infect macrophages (MOI 10:1) for 90min, unless otherwise indicated. Cells were treated with anthrax lethal toxin comprised of 1g/ml protective antigen and 1g/ml lethal factor (List Biological) for 2h¹⁴. Cells were treated with the detergent-based lysis buffer included in the Cytotox 96 Kit (Promega) for 30min. Release of cytoplasmic LDH was determined from triplicate samples using the Cytotox 96 Kit and calculated as 100 (experimental LDHspontaneous LDH)/(maximum LDHspontaneous LDH).

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169L (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

… biological sample (S. aureus) Staph enterotoxin B List Biological Laboratories #122 …

Mixed lymphocyte cultures:

Mouse splenic CD4⁺ T cells were purified with a Stem Cell Technologies negative selection CD4⁺ T cell isolation kit and co-cultured with bone marrow-derived dendritic cells (cultured with GM-CSF for 7 days) at 1:1 ratio with or without Staphylococcal entertoxin B (SEB, 0.05 g/ml, List Biological Laboratories,>95% pure, 14 EU/mg) and in the presence of either IL-21R-Fc blocking agent or control Fc (20 g/ml) for 24 hr; RNA was then isolated. For intracellular staining of IL-21, human CD4⁺ T cells were purified from peripheral blood (Dynabeads Untouched negative selection CD4⁺ T cell kit, Invitrogen) and co-cultured at a 5:1 ratio with monocyte-derived DCs that had been cultured for 6 days in GM-CSF (800 U/ml) plus IL-4 (400 U/ml). At 72 hr, cultures were treated with Golgi Plug for 4 hr and stained 

… Fourteen days post sciatic nerve crush sensory axons that had re-innervated the muscle were traced by injecting 5 µl of 1% CTB (List Biological Laboratories) diluted in saline into the tibialis anterior and medial gastrocnemius muscles using a 10 µl Hamilton syringe and Hamilton needle (NDL small RN ga34/15mm/pst45o ) (Hamilton). …

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

Engineering cell sheets with oral mucosal epithelium cells on transwell:

The isolated epithelial cells were seeded on sixwell plate Transwell (Corning, Tewksbury, MA), at 65,000 cells/cm² in coculture with mitomycin C (MMC)treated NIH/3 T3 feeder cells (Hayashida et al., 2005). OMECs were seeded in the Transwell (Corning, Tewsbury, MA) and were cultured with MMCtreated NIH/3 T3 feeder cells in the following culture media: The culture medium was a mixture of Dulbecco’s modified Eagle’s medium (Sigma, St Louis, MO) and Ham’s F12 (Sigma, St Louis, MO) at a ratio of 3:1 supplemented with 10% fetal bovine serum (ThermoFisher Scientific, Chino, CA), 0.4 g/ml hydrocortisone (Sigma, St Louis, MO), 2 nmol/l triiodothyronine (Sigma, St Louis, MO), 1 nmol/l cholera toxin (List Biological Laboratories, Inc., Campbell, CA), …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

Induction of EAE

EAE was induced by subcutaneous immunization with 50 µg of the myelin oligodendrocyte glycoprotein 3555 peptide (MOG₃₅₅₅; Peptide International, Louisville, KY, USA) emulsified in complete Freund’s adjuvant (Difco Laboratories, Detroit, MI, USA) supplemented with Mycobacterium tuberculosis extract H37Ra (Difco Laboratories) on Day 0. In addition, mice received 250 ng pertussis toxin (List Biological Laboratories, Campbell, CA, USA) intravenously on Day 0 and intraperitoneally on Day 2. Control mice were treated with complete Freund’s adjuvant and saline alone. EAE mice were divided into two groups based on the body weight, and treated vehicle or MR16-1.

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)

General chemicals:

… CT (azide-free) from V. cholerae used for cAMP experiments was purchased from List Biological Laboratories (Campbell, CA, USA) (catalogue no. 100B).

cAMP measurement:

One hundred microlitres of 50 000 cells ml¹ cell suspensions of individual cell lines were added in wells in white-walled 96-well plates (Thermo-Fisher Scientific, catalogue no. 07-200-628) and cultured for 72 h. The wells were washed twice with PBS and then treated with 0.1 nM CT holotoxin at 37°C for 60 min (List Biologicals) in complete induction buffer provided in the cAMP-Glo kit (Promega, catalogue no. V1501). The assay was performed following the manufacturer’s protocol (cAMP-Glo assay; Promega). The luminescence values were obtained using a Synergy Neo microplate reader (BioTek, Winooski, VT, USA).

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

Immunization strategy

Groups of mice (n = 5) were immunized orally in three rounds on days 0 and 1; 14 and 15, and 28 and 29. One hour prior to immunization, food was withdrawn. Mice were gavaged with 200 µl of solution containing 100 µl of 0.3 M sodium bicarbonate buffer (pH 9) and 100 µl Dukoral® with or without α-GalCer (10 μg) (Avanti Lipids) or CT (10 μg) (LIST Biologicals), or with PBS alone. Alternatively, mice were gavaged with 200 µl of 0.3 M sodium bicarbonate buffer. After 20 minutes, mice were gavaged with 200 µl of sterile PBS containing 3 × 108 WCK MS1342 bacteria and 30 µg CTB per mouse with or without α-GalCer (10 μg) (Avanti Lipids) or CT (10 μg) (LIST Biologicals) or with PBS alone. Alternatively, mice were anesthetized with Isoflurane and vaccinated with three SmPill® minispheres which were delivered by loading the minispheres into the silicon tip of a 17 G flexible feeding tube (Agntho’s, Sweden) and 50 µl of 1X PBS adjusted to pH 5.

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

Reagents and diets:

… The adjuvant cholera toxin (CT) was acquired from List Biological Laboratories Inc. (Campbell, CA, USA). …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

… Intraperitoneal (ip) injections of 400 ng pertussis toxin (List Biological Laboratories) were performed on the day of immunization and on day 2. Mice were weighed and scored daily according to following clinical scale: …

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)

Experimental design Oral sensitization of mice to raw and processed cow’s milk

A schematic overview of the experimental design is depicted in Figure 1A. After 1 week habituation, mice (n = 8/group) were sensitized intragastrically (ig) by using a blunt needle with 0.5 mL raw cow’s milk, heated raw cow’s milk or shop milk using 10 µg cholera toxin (CT; List Biological Laboratories) as an adjuvant. …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae