Citations

… Purified recombinant protective antigen (rPA) was obtained from three different sources; 1) Dr. Stephen H. Leppla, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD; 2) List Biologicals, Campbell, CA or …

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

Mice:

C57BL/6 (CD45.2⁺) mice and congenic C57BL/6 (CD45.1⁺) mice were bred in and provided by the animal facility of the Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany. … Cell ablation was achieved by a single i.p. injection of DT (30 ng/g body mass) (List Biological Laboratories) unless stated otherwise. Note that, after birth, mice heterozygous or homozygous for the Ccx-Ckr1-eGFP knock-in allele have no overt thymus phenotype (7).

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

Experimental autoimmune encephalomyelitis (EAE) model:

For analysis of in vivo-generated Th17 cells and Treg cells from an inflammatory site, active EAE was induced and CNS infiltrating leukocytes were obtained as described [32]. Briefly, C57BL/6 mice were immunized by subcutaneous injection in the lower back with 200 µg myelin oligodendrocyte glycoprotein (MOG)3555 (MEVGWYRSPFSRVVHLYRNGK) peptide emulsified at a 11 ratio with complete Freund’s adjuvant containing 150 µg Mycobacterium tuberculosis H37RA (Difco, Detroit, MI). Pertussis toxin (List Biological Laboratories, Campbell CA) was administered on day 0 (200 ng) and day +2 (200 ng) with respect to the immunization day. Only symptomatic mice were used at 14 days after immunization.

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)

Mice and Reagents:

Wild-type C57BL/6 mice were purchased from Jackson Labs. … Control mice received PBS. In vitro stimulation of single-cell suspensions from the lamina propria was performed with a concentration of 200 ng/ml of flagellin. Diphtheria toxin (List Biological Laboratories) was administered every other day at a dose of 10 ng/g starting 6 days prior to injection of flagellin. Mice received a total of three doses.

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

Mice and Reagents:

Wild-type C57BL/6 mice were purchased from Jackson Labs. … Control mice received PBS. In vitro stimulation of single-cell suspensions from the lamina propria was performed using a concentration of 200 ng/mL of flagellin. Diphtheria toxin (List Biological Laboratories) was administered every other day at a dose of 10 ng/g starting 6 days prior to injection of flagellin. Mice received a total of 3 doses.

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

Reagents:

… The PA ligand was purchased from List Laboratories (Campbell, USA). …

Surface modification:

… After washing with HBS for 2 min at a flow rate of 30 m/min, 100 g/ml of PA was introduced and incubated for 30 min with a slow flow rate of 3 l/min for covalent binding to the SAM layer. …

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

Chemicals and Reagents:

PA and LF were purchased from List Biological Laboratories.

Toxin Treatment and Cell Viability Assays:

Mouse macrophages were treated with toxin for 4 h and human B lymphocytes for 48 h. Determination of macrophage viability was done by MTT assay as described in refs. 31, 32, whereas lymphocyte viability was determined by Resazurin (Invitrogen) fluorescence, as described by the manufacturer. Each data point shown in Figs. 1 and and22 represents the average and SD of results from three wells. Cell viability is shown as the percentage of survivors obtained relative to treatment by PA alone (100%). LD₅₀ was calculated for each cell line and normalized to account for variation in toxin potency from the two different lot numbers used, as determined in five and three independent LD₅₀ measurements of a control cell line for the first and second lots, respectively.

Immunofluorescence Microscopy:

PA protein was labeled with Alexa Fluor 647 using the protein labeling kit (A20173; Molecular Probes). We determined by MTT assay that such labeling did not affect the ability of PA/LF to kill macrophages. Fresh serum-free IMDM media was added to cells that contained 1 g/mL PA/Alexa Fluor 647. Cells were incubated for 20 min either at 4 C for PA-binding analysis or at 37 C for determination of PA internalization.

Biochemical Assay of PA Binding and Internalization:

Three million macrophage cells were exposed to 1 g/mL of PA at 4 C for 1 h for binding assay or at 37 C for 20 min for internalization assay. Cells were then washed with PBS solution three times and lysed in RIPA buffer containing a protease inhibitor mixture (Roche). Western blot analysis was performed using mouse monoclonal anti-PA antibody PA (C3) sc-52129 (Santa Cruz Biotechnology), or mouse antiactin monoclonal antibody (Sigma-Aldrich). Chemiluminescence of bands and their relative intensities were revealed using a VersaDoc 1000 instrument (Bio-Rad).

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

Animals and DT Treatment:

…Before behavioral experiments, mice were maintained on standard laboratory chow (5053; Lab Diet) that was available ad libitum. When the Lep^ob/ob;Agrp^DTR/+ and Lep^ob/ob;Pmch^DTR/+ mice were 6 wk old (weighing 2025 g), 8 wk old (weighing 3540 g), or 10 wk old (weighing 4045 g), they were individually housed and switched to another standard chow diet (D12450B; Research Diets) for 7 d before being injected with DT. To ablate AgRP or MCH neurons, DT was injected twice per mouse (i.m., 2 d apart; List Biologicals). Body weight and food intake were recorded daily for several weeks. The DT dose was determined based on body weight at the time of injection, with 50 g/kg for mice weighing <40 g and 40 g/kg for mice weighing >40 g. …

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

Materials and Methods:

… CTB was visualized by sequential incubations in a primary goat anti-CTB antibody (List Laboratories; 1:30,000 in 0.3% Triton X-100 in PBS). This antibody was raised against purified choleragenoid and does not result in labeling following preabsorption of the antibody with excess concentration of choleragenoid (Stocker et al., 2006), and no labeling is seen in material in which a CTB injection has not been performed (Kubke et al., 2004). Incubation in the primary antibody was followed by …

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

Histology:

… Every third section of cortex was processed for cytochrome oxidase (CO; Wong-Riley, 1979b), to reveal the V1V2 border and the CO-dense stripes of V2, and another series of every third section was processed for CTB by using an antibody and the histological procedures described in Bruce and Grovfova (1992). See Table 2 for the CTB antibody source. The CTB antibody failed to stain cells in tissue from animals without CTB injections. A third series was processed for myelin, or set aside. Every third section of the thalamus was processed for CO to help determine lateral geniculate nucleus (LGN) and pulvinar borders. A second series of every third sections was processed for CTB, and the third series was set aside.

… Table 2. Antibody Used in This Study. Antigen, Immunogen, Manufacturer, Dilution. Cholera toxin subunit B (CTB), Purified CTB isolated from Vibrio cholerae, List Biological Laboratories (Campbell, CA), goat polyclonal #703, 1:5,000. …

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