Citations

EAE model:

… On days 0 and 2 of disease course, mice were also injected IP with 375ng of pertussis toxin (List Biological Labs, Campbell, California, USA). Mice were sacrificed at peak disease, PID 16 and late stage, PID 42. For controls the same protocol was used but mice were immunized with CFA without peptide. …

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)

… iDTRwt/ki mice were injected for three consecutive days intravenously with 0.5 g of unnicked Diptheria Toxin from Corynebacterium diphtheria (List Biological Laboratories) diluted in sterile PBS. On day 4 niche cell depletion was analyzed by flow cytometry …

• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae

… pertussis toxin was purchased from List Biological Laboratories (Campbell, CA); …

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)

… For Foxp3+ cell depletion in DEREG mice, diphtheria toxin (#150; List Biological Laboratories, Campbell, Calif) was administered intraperitoneally 12 hours before each Cl2 exposure at a concentration of 40 ng/g body weight. …

• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae

Experimental DesignTolerance Induction, Sensitization and Challenges:

… Following this oral tolerance induction period, mice were sensitized i.g. once a week for five weeks to the hens egg protein ovalbumin (OVA; 20 mg/0.5 mL PBS; grade V; Sigma-Aldrich, Zwijndrecht, The Netherlands) while using 10 µg cholera toxin (CT; List Biological Laboratories, Campbell, CA, USA) as an adjuvant (days 0, 7, 14, 21, 28; n = 8/group). Sham-sensitized control mice (n = 6) received CT alone (10 µg/0.5 mL PBS). …

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

Human PBMC Synergy Assays:

Human peripheral blood mononuclear cells were isolated using single step FicollPaque method, then washed three times to remove platelets and resuspended in RPMI-1640 supplemented with 10% HI-FCS, 2 mM L-glutamine, 2.5 mM HEPES + penicillin/streptomycin) at 10000 cells/well in 96- well flat bottom plate. Several pilot experiments (minimum of four different donors, and typically six) were performed using a full dose response of ligand to define the range of the EC10 to EC30 of MDP (Invivogen TLRL-MDP) and Pam2CSK4 (Invivogen TLRL-pca2-1), respectively, as it was determined that this was the concentration needed to reliability and consistently demonstrate synergy using both ligands. Typically, three concentrations of each of the two ligands were evaluated in a pairwise matrix to account for minor variations between donors, and also a full doseresponse was assessed to compare to the average of all donors tested. Compounds were preincubated for 30 min, and then ligands were added, and plates were incubated for 4 h at 37 C with 5% CO2. Supernatants were assessed for human proinflammatory cytokines using an MSD four plex kit (K15025C-4). Raw data were converted to cytokines concentrations using a four-parameter curve fit with 1/y2 weighting as recommended by the manufacturer using PerkinElmer Workout software. Cytokine values were then graphed into dose response curves using Sigma Plot software and an IC50 was determined for individual and composite graphs. The synergistic response was compared to the response of each of the ligands alone for each donor. A similar approach was also used for MDP and Ultrapure LPS (List Biologicals 421), MDP and Flagellin (Invivogen TLRL-stfla), and last MDP and IL-1ineb (Biolegend 57006). 

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

… EAE was induced in C57Bl/6 mice (female, aged 6-8 weeks, Charles River, Wilmington, Massachusetts) by intraperitoneal injection of 250 ng of pertussis toxin (List Biological Laboratories, Campbell, California) and subcutaneous injection of 300 µg of MOG35-55 in …

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)

… OVA (1 mg) was given by oral gavage (with each mouse receiving 10 µg of cholera toxin (CT); List Biologicals, Campbell, CA) twice a week for 4 weeks, and various monosaccharide-conjugated forms of OVA (OVA-Glu, OVA-Man, OVA-Fru, and OVA-Rib) and the control OVA were administered orally thrice a week for 4 weeks, as described in Figure 1.

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

… After LPA pretreatment, the cells were stimulated for indicated time with 100 ng/ml ultrapure smooth LPS from Escherichia coli O111:B4 referred to as LPS (List Biological Laboratories, Campbell, CA, USA) …

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

Reagents:

rCTxB List Labs Cat# 103B

Tetanus toxoid List Biological Laboratories Cat# 191A

ELISA:

… Additional ELISA assays were performed by coating plates with 1 µg/mL recombinant protein antigens CtxB (Kauffman et al., 2016) and influenza HA-antigen (A/California/7/2009, eEnzyme). Minimum effective concentration was determined as the minimal concentration of mAb at which an OD signal was detectable 3-fold above the background signal measured by a negative control.

To determine serum titers against tetanus-toxoid protein, ELISA plates (ThermoScientific) were coated with 0.5 µg/mL of recombinant Tetanus toxoid (List Biological Laboratories) diluted in PBS and incubated overnight at 4°C. …

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt
• Product #191 – Tetanus Toxoid from Clostridium tetani