Citations

Cell transfection and infection:

Mycoplasma free-ZF4 cells (ATCC CRL-2050), established from 1-day-old zebrafish embryos, were grown at 28C in Dulbeccos modified Eagles medium (DMEM)/F12 (Gibco) medium supplemented with 10% fetal bovine serum (FBS; Gibco) in an atmosphere containing 5% (v/v) CO₂. These cells were infected by replacing the culture medium with Opti-MEM (Gibco) and adding E. piscicida at a multiplicity of infection (MOI) of 50. For CTB (List Biological Laboratories) treatment, Pam3CSK4 (InvivoGen) primed cells were stimulated with 20mg/mL CTB plus 1mg/mL ultrapure LPS (E. coli O111:B4, InvivoGen)^22,41. For inhibitor assays, the cells were pretreated with Z-WEHD-FMK (ApexBio), Z-YVAD-FMK (ApexBio), Ac-LEVD-CHO (Sigma), Z-VAD-FMK (Beyotime Biotechnology), Ac-DEVD-CHO (Beyotime Biotechnology), or 10mM glycine (Sigma), respectively, for 30min before being exposed to E. piscicida at an MOI of 50.

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

Cholera toxin subunit B (CTb) Antiserum Cholera toxin B subunit List Biological Laboratories; Goat; Polyclonal CAT No. 703; Lot No. 7032A7 …

• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

Materials and Methods:

… Cholera toxin (CT) was purchased from List Biological Laboratories. …

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

… PTX (Pertussis toxin) List biological laboratories, Campbell, CA, USA …

Mouse immunization with MOG35-55 and EAE induction:

AhRDC , AhRTreg , AhRAstro , AhRCD4 and AhRfl/fl control mice were actively immunized with a subcutaneous (s.c.) injection of 50 µl of MOG35-55 emulsified in complete Freunds adjuvant (CFA, composition detailed in table 3) per groin and per axilla (200 µg MOG35-55/animal). The s.c. inoculation was accompanied by an intraperitoneal (i.p.) injection of 400 ng of pertussis toxin (PTX) on the day of immunization and two days 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)

… PA 63 was prepared from PA 83 using furin [52] or purchased from List Biological Laboratories, Inc. (Campbell, CA, USA) in the purified form …

• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis

… On Day 10 all animals were anesthetized with isoflurane, mounted in a stereotaxic instrument, and received a unilateral injection of the retrograde tracer cholera toxin B (CTB; List Biological Laboratories; 1% solution) into the BLA …

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

Tracer injection protocol.:

As previously described [21], glass micropipettes … were filled with 1% desalted CTb (List Biological Lab.) …

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

Materials:

Hinute-AM was kindly provided by Fuji Oil Co., Ltd. We used smooth-type LPS from E. coli O55:B5 (List Biological Laboratories, CA, USA) as …

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

… Additionally, the mice receive a peritoneal injection of 200 ng of pertussis toxin (List Biological Laboratories, Campbell, CA, USA) dissolved in PBS …

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)

Protease assay:

Proteolytic activity was determined, as described earlier [22Kaman WE, Hulst AG, van Alphen PT, et al. Peptide-based fluorescence resonance energy transfer protease substrates for the detection and diagnosis of Bacillusspecies. Anal Chem. 2011;83:25112517.[Crossref], [PubMed], [Web of Science ], [Google Scholar]], by incubating 16 M FRET-peptide with varying concentrations of PA₈₃ or LF (both List Laboratories) in HEPES buffer (20 mM HEPES, 0.05% Tween-20, pH 8.2) using blackwell clear bottom 96-well plates (Corning). …

Cleavage characteristics of PA₈₃

20 g/mL PA₈₃ was incubated with 16 M FRET-peptide PEK-054 (FITC-NleKKKKVLPIQLNAATDK-KDbc) [20Cummings RT, Salowe SP, Cunningham BR, et al. A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease. …

LC MS/MS:

Prior to LC MS/MS analysis 10 g PA₈₃ was digested with 0.5 g trypsin (Difco, BD Biosciences) for 3 hr at 37C. The trypsinized sample was analyzed by MS/MS using the data dependent LC-auto MS/MS mode. …

SDS-PAGE:

PA₈₃ protein (2 g) was mixed 1:1 with 2x Laemmli sample buffer (Biorad), incubated for 5 min at 95C and loaded onto a 10% SDS-PAGE gel. After electrophoresis for 1 hr at 22 mA (max. 160 V) the gel was stained overnight with PageBlue staining solution (Fisher Scientific) and washed two times to visualize bands.

Specificity testing PA₈₃

20 g/mL PA₈₃ was incubated with 15 g/mL B. anthracis protective antigen antibody-[HRP] (-PA, Novusbio, BAP0102), 15 g/mL human IgG antibody-[HRP] (-human IgG, Sigma Aldrich, A0170) or PBS (solvent of the used antibodies) in HEPES buffer for 5 hr at room temperature. Next, 1 L PEK-054 (800 M) was added to the reactions and increase in fluorescence was measured at 37C for 60 min as described above.

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