Bacterial Toxin Research Citations

Materials:

Escherichia coli O111 B4-derived LPS was obtained from List Biological Laboratories, Inc. (Campbell, CA, USA).

Northern blot analysis and Cox2 mRNA inhibition:

The procedure employed was similar to that reported previously (14). Briefly, 106 cells were placed in Falcon 5-cm-diameter dishes (Becton Dickinson Labware, Franklin Lakes, NJ, USA) and pre-treated for 30 min with each of the phenol-related compounds at a concentration of 106 M, 105 M or 104 M. They were then incubated in the presence or absence of LPS (100 ng/ml), and their total RNA was prepared 3 h later by the acid guanidine-phenol-chloroform procedure (15). …  Statistical analyses were performed using Students t-test. Inhibition of LPS-stimulated Cox2mRNA expression byp-cresol, p-cresol dimer, pHA, pHA dimer and BHA in RAW264.7 cells was carried out as follows: The cells were pretreated for 30 min with or without addition of 105Mp-cresol, pHA,p-cresol dimer, pHA dimer or BHA, and then incubated with or without LPS at 100 ng/ml for 3 h. Total RNA was prepared and Cox2 mRNA expression was confirmed by northern blot analysis.

Preparation of nuclear extract and microwell colorimetric Nfb assay:

Nuclei were extracted and prepared for the microwell colorimetric Nfb assay. In brief, the cells in 5-cm-diameter Falcon dishes (106cells per dish) were pretreated for 30 min with or without the indicated doses of the compounds, and then treated with LPS at 100 ng/ml for 1 h. …Inhibition of LPS-stimulated Nf-b activation byp-cresol,p-cresol dimer, pHA, pHA dimer or BHA in RAW264.7 cells was carried out as follows: The cells were pretreated for 30 min with or without addition of 105 M p-cresol, pHA, p-cresol dimer, pHA dimer or BHA, and then incubated with or without the LPS at 100 ng/ml for 1 h. Nuclear extracts were prepared and used in a TransAM (Active Motif) ELISA-like assay to quantify the Nfb p50, p52, p65 and RelB DNA-binding activity. …

• Product #201 – LPS from Escherichia coli O111:B4

… To determine whether antisera induced by CTB or CTBCOMP fusion protein with or without heat treatment inhibit CT from binding its receptor GM1, 100 µl of the serially diluted antisera were incubated with 100 µl of CT (0.4 µg/ml in PBS, List Biological Laboratories, Inc …

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

Murine experimental autoimmune encephalomyelitis (EAE):

…Active induction of EAE was performed with a subcutaneous injection of each mouse with 200µg of myelin oligodendrocyte glycoprotein (MOG) 3555 peptide… Two intra-peritoneal (i.p.) injections of Bordetella pertussis toxin (PTX; List Biological, Campbell, CA) at 200 ng pe mouse was given at the time of 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)

Inhibition of Rho GTPase activity:

To study cellular phenotypes independent of GTPase activation, cells were treated with either Clostridium difficile toxin B (TcdB) or C3 transferase to irreversibly inactivate either RhoA, Rac and Cdc42 or RhoA, respectively. Cells were treated wither with 200 ng/ml TcdB (List Biologicals) or 1 g/ml cell-permeable C3 (Cytoskeleton) for 4 hours. Attachment experiments were carried out immediately after toxin treatment.

• Product #155 – Toxin B from Clostridium difficile

Chemicals:

… cholera toxin was from List Biological Laboratories, Inc. (Campbell, California, USA) …

Mouse models of cholera and intestinal fluid absorption assays

To investigate the in vivo effect of diclofenac on CT– and V. cholerae-induced intestinal fluid secretion, mice (30–35 g, ICR strain; The National Laboratory Animal Center, Salaya, Nakornpathom, Thailand) were fasted for 24 h before experiments. After anesthesia by an intraperitoneal injection of thiopenthal sodium (50 mg/kg), an abdominal incision was made and 3 closed ileal loops (2–3 cm in length) were made by ligations and then instilled with 100 µl of phosphate-buffered saline (PBS) or PBS containing CT (1 µg) or V. cholerae (classical O1 569B strain of V. cholerae at 107 CFU/loop). This strain of V. cholerae was used since it has been known to produce large amounts of CT and cause consistent intestinal fluid secretion in adult mouse closed-loop models [17]. Body temperature of mice was maintained at 36–37°C for the entire period of operation using heating pads. After abdominal closure by sutures, mice were intraperitoneally administered with DMSO (control) or diclofenac (30 mg/kg), and allowed to recover from anesthesia. Four hours (for experiments using CT) or 12 hours (for experiments using V. cholerae) later, mice were anesthetized again, abdomen was opened and ileal loops were removed for measurements of intestinal fluid secretion from loop weight/length ratios. Then, mice were euthanized with an injection of thiopenthal sodium (150 mg/kg). …

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

… For SL administration, the HA1 protein was mixed with wild-type cholera toxin (wtCT) (List Biological Labo- ratories, Inc., USA) as adjuvant. …

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

Generation of Abs using vaccine candidates.

… Each mouse was immunized i.n. with 50 µl of 2×10 7 PFU of inactivated PR8 mixed with 2 µg of cholera toxin (List Biological Laboratories, Inc., Campbell, CA). The PR8-primed mice were then boosted by a second immunization using the same procedure. …

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

Chemical reagents and induction of skin inflammation:

Cholera toxin was purchased from List Biological (Campbell, Calif). …

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

Analysis of regeneration of dorsal column fibers:

The slices were incubated for 1 hour in blocking solution ( 5% donkey serum + 0 , 3% Triton X – 100 in 0 , 1M PB ) at room temperature and overnight at 4C with goat anti-CTB (List Biological Laboratories Inc 703, 1:30000 ) in blocking solution and put 2 hours in anti-goat biotinylated IgG (Vector BA – 5000 , 1:200 ) in blocking buffer. …

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

Reagents and Abs:

Ultra-pure LPS (Escherichia coli 0111:B4) was obtained from List Biologicals (Campbell, CA).

Animal experimentation:

…Mice were anesthetized using a mixture of isourane and oxygen bled in at 3.5 l/min.  LPS (List Biologicals)dissolved in DNase/RNase-free distilled water (1 mg/ml) was instilled i.p. at a concentration of 1.5 or 5 mg/kg. Animals were sacriced 18 or 48 h after LPS. …

Cell isolation and treatment:

… Selected cells were then treated with LPS (100 ng/ml) for the durations described in the gure legends.
Small interfering RNA

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