Citations

Mice and EAE induction:

…In most experiments IL-12R2^/ and WT mice were immunized for EAE induction with 200 µg PLP₁₃₉₁₅₁ (GenScript) emulsified in complete Freunds adjuvant (CFA) containing 3 mg/ml Mycobacterium tuberculosis (H37Ra; Difco Laboratories). In addition, mice were given 200 ng pertussis toxin (List Biologicals Laboratories) i.p. on days 0 and 2 post-immunization (p.i.). …

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:

… emulsified with complete Freund’s adjuvant (CFA) containing 500 µg of Mycobacterium tuberculosis H37RA (BD Biosciences) on day 0 and were intraperitoneally (ip) injected with 500 µg of pertussis toxin (PTX; List Biological Laboratories, Campbell, CA) on days 0 and 2 …

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:

…EAE was induced by s.c. immunization at four sites on the back with 100 mg rhMOG emulsied in CFA containing 5 mg/ml mycobacteria… On days 0 and 2, mice were injected i.p. with 300 ng pertussis toxin (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)

Retrograde tracing and histology:

… We anaesthetized the Pdyn-ires-Cre; Z/EG and the Pdyn-ires-Cre; R26-loxSTOPlox-L10-GFP mice with ketamine (100 mg kg¹, ip) and xylazine (10 mg kg¹, ip) and stereotaxically unilaterally microinjected 69 nl of the retrograde tracer cholera toxin subunit B (CTB; List Biological, Campbell, CA, USA; 0.2% in saline) into the MCPO/SI (AP: 0.46 mm, DV: 5.3 mm, ML: 1.2 mm) and these animals were then killed 1 week later. …

For the retrograde tracing study, we first surveyed the entire brain using light microscopy. We incubated sections overnight in goat anti-CTB primary antiserum (1:10,000; List Biological Laboratories; catalogue no. 703; lot no. 7032A7) and …

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt
• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

Immunizations and EAE evaluation:

On day 0, mice were immunized subcutaneously with 50 µg of a myelin oligodendrocyte glycoprotein peptide (MOG₃₅₅₅) emulsified 1:1 volume in CFA supplemented with 4 mg/mL Mycobacterium tuberculosis (H37Ra; Difco Laboratories, Detroit, MI, USA). Mice were additionally injected intraperitoneally on days 0 and 2 with 250 ng of pertussis toxin (List Biological Laboratories, Campbell, CA, USA). …

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)

In Vivo Depletion and Transfer of Donor and Recipient Leukocyte Subsets:

Hematopoietic cells were depleted as previously described…by lethal irradiation (2  6.5 Gy) of donor mice 7 days before heart allograft procurement. B6.CD11c-DTR, BALB/c.CD11c-DTR, and CB6F1.CD11c-DTR donors and C57BL/6 (BL/6.CD11cDTR) chimeric heart graft recipients were treated with i.p. 32 ng/g DT (List Biological Laboratories) on days 5, 3, and 1 (donors) or thrice weekly in recipients.

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

TT-specific human T cell response:

For in vitro TT stimulation, autologous DCs were co-cultured with CFSE-labeled purified human T cells at different ratios in the presence of 50 ng/ml TT (List Biological Laboratories) for 1014 d.

• Product #190 – Tetanus Toxin from Clostridium tetani

Induction of active EAE and immunizations:

For EAE induction, mice were immunized subcutaneously with 100 g MOG₃₅₅₅ (CS Bio Co.) emulsified in CFA (made with 5 mg/ml heat-killed Mtb H37Ra [BD] in incomplete Freunds adjuvant [BD]). PTX (List Biological Laboratories) or mutant PTX (mPTX; mutated at two positions in the S1 subunit [R9K and E129A]; List Biological Laboratories) was injected i.p. (300 ng) on days 0 and 2.

Author did not specify which List Labs Pertussis Toxin was utilized.  List Labs provides Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer and Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free).

• Product #184 – Pertussis Toxin Mutant

Materials and Methods

Anthrax lethal factor (Product #169), and the chicken IgY polyclonal anti-LF antibody (Product # 769A) are products of List Biological Laboratories,Inc.…

Sample Preparation:
Stock solutions of the fluorogenic substrate, MAPKKide® Plus, were 1.25 mM in DMSO based on the peptide content
determined by elemental analysis. The substrate was diluted in assay buffer: 20 mM HEPES, pH 8.0 containing 0.1% Tween-20. For the microplate
assays, the LF was dissolved in neat bovine plasma and diluted 1:10 in assay buffer. For the HPLC assays, the LF was added to neat bovine plasma and not diluted.

LF Activity Assays:
Microplate reader: Assays were performed on a SPECTRAmax GEMINI XS fluorescence microplate reader (Molecular Devices). The
cleavage reaction was initiated by addition of the substrate, MAPKKide® Plus. The concentration was optimized to minimize background fluorescence while maintaining measureable cleavage. For all experiments the time-dependent increase in fluorescence was monitored at 37°Chourly for 5 or 6 hours followed by an additional 18 to 18.5 hr overnight incubation at ambient temperature. The excitation wavelength was set to 368 nm and emission to 452 nm with a cutoff filter at 435 nm.

HPLC:
The C8 Starwell Maxi Nunc-Immuno Module Plates were coated with 150 μl of a 10 μg/ml solution of a chicken affinity purified
polyclonal IgY antibody to anthrax lethal factor (List Prod # 769A). Plates were incubated with the IgY overnight at 2-8°C and washed three times with 0.1M Glycine-HCl, pH 2.5. This wash was included to liberate residual LF retained after the affinity purification of the antibody and was necessary to minimize the background. After 6 washes with PBS containing 0.05% TWEEN-20 (PBST), the anti-LF coated wells were exposed to 300 μl of a series of LF concentrations in neat plasma. The plates were incubated at 22°C for 2 hours. Plates were then washed 6 times with PBST and 250 μl of 1.25 μM MAPKKide® Plus was added. The reaction was allowed to proceed for 2, 3.5, and 5 hours at 37°C and overnight at ambient temperature. At each time point 200 μl of the reaction mixture was removed from replicate wells and placed in HPLC sample vials. HPLC was performed using a Zorbax Eclipse Plus C18 reverse phase column, 4.6 x 150 mm (Agilent) and a guard column containing the same resin in a Varian ProStar HPLC system (Agilent). Solvent A was 0.1% TFA in water and solvent B was 0.1% TFA in acetonitrile. The 16 minute HPLC method was as follows: 25% B for 0.75 minutes; 25 to 45% B in 4.75 minutes; 45 to 100% B in 0.75 minutes; 100% B for 3.75 minutes; 100 to 25%B in 0.67 minutes and 5.34 minute equilibration with 25% B. The column effluent was monitored using an Hitachi fluorescence detector with excitation set to 350 nm and emission at 450 nm to detect the free coumarin fluorophore cleaved from MAPKKide® Plus. The injection volume was 20 µl. The 7-amido-4-methylcoumarin (AMC) peak retention time was 4.8 minutes.

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
• Product #769B – Anti-Lethal Factor from B. anthracis (Chicken lgY)
• Product #532 – MAPKKide® Plus (AMC) Specific Substrate for Anthrax Lethal Factor

Oral immunization:

Aged mice with or without AMSC transfer and young adult mice were immunized three times at weekly intervals with oral doses of 1 mg of OVA (Fraction V; Sigma, St. Louis, MO) and 10 µg of CT (List Biological Laboratories, Campbell, CA) in PBS…

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