Bacterial Toxin Research Citations

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)

EAE:

… Mice received iv injection of 200 ng pertussis toxin (List Biological Laboratories, Campbell, CA) on days 0 and 2 post immunization (pi). Mice were weighed and evaluated for neurological disability daily. …

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 procedures:

Adult BALB/c WT mice were purchased from Jackson Laboratory (Bar Harbor, ME). Ad5 vectors were injected intramuscularly (IM, into the tibialis anterior of the right hindlimb, total volume 25 l) into 8 weeks old male mice after performing proper anesthesia with isofluorane. A total of 1 10¹⁰ vp per mouse was administered IM. Number of animals used for each experiment is specified on corresponding figure legend. Toxin A challenge experiments were performed at 14 dpi as previously described [14] by injecting 300 ng of freshly reconstituted toxin A (List Biological Laboratories Inc., Campbell, CA …

• Product #152C – Toxin A from Clostridium difficile

Spinal cord and brainstem:

… Alternating horizontal sections were reacted for cytochrome oxidase, enabling clear demarcation of the gray and white matter. The other sections were incubated with primary goat anti-CTB (1:4,000) (#703; List Biological Laboratories) diluted in Tris-buffered saline containing 0.25% (vol/vol) Triton X-100 (Sigma) and 2.5% (vol/vol) normal rabbit serum (Millipore) to visualize the forelimb primary afferents and terminal fields. The brainstem was separated, blocked, and left in …

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

Cell culture assays:

… Ultra Pure lipopolysaccharide (LPS) from E. coli serotype O111:B4 was obtained from List Biological Laboratories (# 421, List Biological Laboratories, INC., Campbell, CA, USA). …

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

Epicutaneous Immunization.

Immunization was typically on the ear skin, without prior treatment. In some experiments (specified in the text) the flank skin was used, in which case mice were shaved (under anesthetic) at least 2 d before immunization (16). Immunization followed the scheme described in SI Appendix, Fig. S1. Briefly, mice were anesthetized and the skin site hydrated with water (15 min), then OVA protein (500 μg in 25 μL of PBS; Sigma-Aldrich) applied topically for 20 min. After washing with water and air drying, the indicated adjuvants were applied to the same site for 20 min. Unless otherwise indicated, the following doses of adjuvants were used: CT (List Biological Laboratories), 100 μg; CpG 1826 (Invivogen) 500 μg; Poly I:C (Amersham) 200 μg; LPS (Sigma) 10 μg; and imiquimod (3M) 50 μL of 1% cream. The immunization site was extensively washed with water and allowed to dry before the animals recovered. In the case of recombinant CT and CT(mut) used for the OVA–CT fusion studies, 10 μg of CT [or CT(mut)] was used. For sortase fused OVA–CT/CT(mut), the immunization involved a single application step. In titration experiments we found similar adjuvant effects using either 10 or 100 μg of commercial CT for EPI on ear skin, but for experimental consistency data with 100 μg CT are shown throughout. The control mice received PBS in place of antigen or adjuvants, as indicated.

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

… For direct ELISA analysis of the peptide binding affinity to a recombinant PA protein, 83 kDa PA (PA 83 ; List Biological Laboratories, Campbell, CA, USA) was labeled directly with 44 kDa horseradish peroxidase enzyme (HRP) using EZ-Link plus activated peroxidase …

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