Citations

Adjuvants:

B. pertussis toxin and DNA containing unmethylated CpG-dinucleotide were purchased from List Biological and Operon, respectively. NE was made at the Centers for Disease Control and Prevention (CDC) in Atlanta, GA. This adjuvant is composed of a water phase (PBS, Tween 80) and oil phase (Squalene with or without -tocopherol) at a 4:1 ratio. The nanoemulsion was prepared by passing the mixture through M-110 PMicrofluidizer (Microfluidics) under a pressure of 18,000 psi. …

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)

Animal models and ethics statement:

… All immunizations were performed by instilling 20 µl of a mixture of PBS, 1 µg of cholera toxin (CT; List Biologicals, Campbell, CA) and either 4 µg of the indicated purified pneumococcal protein or 100 µg of WCC as immunogens onto the nares of gently restrained unanesthetized mice. Control animals were immunized with CT mixed in PBS alone to control for any nonspecific effect of the adjuvant alone. Animals were immunized intranasally twice at one-week intervals. …

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

… After this, cells were preincubated for 30 min with 50 ng/mL of IL-10, 5PEGIL-10 or 20PEGIL-10 in 0.1 mL FBS-free medium containing 0.5% normal mouse serum. At t = 0, 25 ng/mL of LPS (E. coli, List Biological Laboratories, INC., USA) was added to the wells. …

Author did not specify which List Labs LPS product was utilized in their research.  List Labs provides the following LPS products:  https://listlabs.com/product-information/lipopolysaccharides/

Materials:

The following TLR agonists were purchased: smooth LPS from E. coli O55:B5 (List Biologicals) …

Macrophage culture and cytokine assays:

BMDMs were washed three times in serum-free medium, followed by incubation overnight with HMGB1, with or without hemoglobin, or with different TLR agonists as desired at indicated concentrations.NOTE:  At the time this paper was written, Product #203 (5mg – LPS from Escherichia coli O55:B5) was utilized.

NOTE:  At the time this paper was written, Product #203 (5mg – LPS from Escherichia coli O55:B5) was utilized.

Product #203 (5 mg – LPS from Escherichia coli O55:B5) is no longer sold.

Product #203A (2.5 mg – LPS from Escherichia coli O55:B5) is available for purchase.

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

Cell culture:

Bone marrow-derived macrophages (BMDM) were prepared by harvesting marrow from femurs and tibias of 129+/+ and Nlrp1b2/2 mice. Marrow was cultured for 7 d in IMDM containing 15% L cell-conditioned medium, 10% FBS, 2 mM L-glutamine, 150 U/ml penicillin, 150 mg/ml streptomycin, and 57.2 mM 2-ME (all from Life Technologies). BMDM were incubated in OPTIMEM plus Glutamax (Life Technologies) containing varying concentrations of LT (List Biological Laboratories, Campbell, CA) for 4 h, and cell viability was assessed with the WST-1 reagent (F. HoffmannLa Roche, Basel, Switzerland). Peritoneal macrophages were elicited from wild-type, Nlrp1b2/2, Nlrp32/2, and Casp-12/2 mice with an i.p. injection of 4 ml 4% thioglycollate in PBS. Seventy-two hours after injection, the peritoneal cavity was lavaged with DMEM, and cells were plated and incubated for 1 h. Adherent cells were then washed twice with cold PBS and cultured in DMEM containing 10% FBS, 2 mM L-glutamine, 150 U/ml penicillin, 150 mg/ml streptomycin, and 57.2 mM 2-ME (all from Life Technologies). For LT treatments, macrophages were primed overnight with 100 ng/ml ultrapure LPS (Invitrogen Life Technologies), washed with PBS, and incubated with OPTIMEM plus Glutamax containing either PBS or 1 mg/ml LT for 6 h. …

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 #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

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

Materials:

… Ultrapure LPS (TLR4 ligand) isolated from Salmonella minnesota R595 was obtained from List Biological Labs (Campbell, CA). …

Cell culture and ELISA:

… Monocytes were isolated with .95% purity, based on CD14 positivity in ow cytometric analysis and were maintained in RPMI 1640 supplemented with 10% FCS. Cells were incubated with TLR ligands PAM2, PAM3, and LPS, or with M. tuberculosis whole cell lysates for 24 h. …

• Product #434 – ULTRA PURE LPS from Salmonella minnesota R595 (Re)

EAE immunization:

Mice were immunized s.c. over four sites on the ank with 100 ml of an emulsion containing 200 mg myelin oligodendrocyte glycoprotein (MOG) 3555 (CS Bio) in PBS and an equal volume of CFA (containing 200 mg heat-killed Mycobacterium tuberculosis, Jamaica strain). Mice also received 200 ng pertussis toxin (List Biological Laboratories) injected i.p. at the time of immunization and 48 h later. …

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 induction:

For active EAE, B6 mice were s.c. immunized with 100 mg MOG3555 peptide (MEVGWYRSPFSRVVHLYRNGK) (Biopolymers Laboratory, Harvard Medical School) emulsied in CFA solution (4 mg/ml mycobacteria in IFA). On the day of immunization and 2 d after, mice received 200 ng pertussis toxin (List Biological Laboratories) in 100 ml PBS i.v. …

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)

… A glass micropipette (20-30 m id) filled with 1% low salt CTB (List Biological Laboratories, Campbell, CA) in distilled water was stereotaxically lowered into part of the left retrosplenial cortex or presubiculum, where BDA-labeled terminals were densely distributed in the …

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

EAE:

… Intraperitoneal injections of 500 ng pertussis toxin (List Biological Laboratories, Campbell, CA) dissolved in 200 µL PBS were administered immediately after the first MOG injection on Day 0 and 48 hours later. …

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)