Bacterial Toxin Research Citations

Mouse studies

EAE was induced in C57Bl/6J mice (Jackson Laboratory) and SJL/J mice, aged 8–15 weeks. Complete Freud’s Adjuvant with 4mg/mL crushed, deactivated mycobacterium tuberculosis and 1mg/mL myelin peptide [MOG 35–55 (C57Bl/6) or PLP139-151 (SJL/J)] were emulsified and injected subcutaneously above shoulders and hips of anesthetized mice, 50 μL per injection site for a total of 200 μL emulsion. For studies using C57Bl/6J mice, pertussis toxin (List Labs, 50μg/mL) in 200μL PBS was diluted to 200–400 ng depending on lot potency and injected intraperitoneal (i.p.) on the day of immunization (day 0) and 2 days postimmunization (day 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)

• Product #162L – Shiga Toxin 2 from Escherichia coli

Shiga toxin work

Shiga toxin 2 (Stx2) was obtained commercially from the USA through List Biological Laboratories Inc. (product #162 lot 1621A1). Stx2 was chosen as it is known to be more cytotoxic in mice than Stx1; and because epidemiological data from human studies have demonstrated that Stx2 producing STEC have a stronger association with more severe disease.22 All work involving the toxin was performed in a biological cabinet under category 3 facilities. Prior to any toxin work commencing, a ‘control of substances hazardous to health regulations’ (COSHH) risk assessment was completed which was submitted and reviewed by the biological and genetic safety committee.

Upon receipt of Shiga toxin, the lyophilized powder was reconstituted with 100μL distilled water and gently mixed by inversion in-keeping with the manufacturer’s instructions. This resulted in a stock concentration of 10μg/100μls which was aliquoted into 20μL stock eppendorfs and stored at −20°C until use. The toxin was assessed for purity on 12% SDS-PAGE and was found to have two major bands at molecular weights of 33 kDa and 8 kDa corresponding to A and B toxin subunits, respectively. Purity was >98% and the endotoxin content determined on kinetic chromogenic LAL assay at List Labs was <90EU/mg. A dose of 10 ng/g of Shiga toxin diluted in sterile normal saline 0.9% (Baxter) was given to each mouse via intraperitoneal injection. All in vitro Shiga toxin cell treatments were performed on differentiated cells at a dose of 0.1 ng/ml.

2.1. Materials

E. coli Shiga toxin 1 Stx1 enterotoxin List Laboratories #161 0.2, 1, 2 μg/ml
Clostridium difficile Toxin B ToxB enterotoxin List Laboratories #155B 0.2, 1, 2 μg/ml
Clostridium difficile Toxin A ToxA enterotoxin List Laboratories #152B 2 μg/ml

2.4. Exposure to proteins

… Four IEC monolayers/treatment group were exposed to (−) control media alone, (+) control Clostridium difficile Toxin A, (+) control TritonX-100, individual test proteins at three different concentrations, or a mixture of the two-component insecticidal protein (AfIP-1A + AfIP-1B) at three different concentrations. The concentrations of the proteins added were based primarily on the solubility of the protein and/or the amount that were commercially available. …

• Product #161 – Shiga Toxin 1 from Escherichia coli
• Product #155 – Toxin B from Clostridium difficile
• Product #152C – Toxin A from Clostridium difficile

EAE induction

Experimental autoimmune encephalomyelitis (EAE) was induced in eight week-old female C57BL/6 mice via immunization with myelin oligodendrocyte glycoprotein (MOG)35–55 (PolyPeptide Laboratories). 200 μg MOG35–55 was dissolved in complete Freund’s adjuvant containing 400 μg of Mycobacterium tuberculosis (231141, Difco) and injected subcutaneously in 0.2mL volume per mouse. Pertussis toxin (181, List Biological labs Inc) was administered via intraperitoneal injection after the MOG 35–55 injection and two days later at 75ng and 200ng per mouse doses, respectively.

• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)

… Mice were injected intraocularly with the cholera toxin B fragment (CTB, 1% in 2 µL; List Biological) conjugated with Alexa-555 72 h before sacrifice. …

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

… injection of antigen emulsion at a dose of 0.75 ml/kg into its footpad, followed by an auxiliary injection of 8 µg/kg diluted in 0.5 ml pertussis toxin (List Biological …

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)

2.3. Antigen-induced EAE (aEAE) models

The aEAE models were established in our previous study.[4] To actively induce disease in mice, we injected a 200 mL emulsion containing 200 µg of myelin oligodendrocyte glycoprotein peptides 35–55 (MOG35–55; amino acids 35–55 of bovine MOG; Sigma-Aldrich, St. Louis, MO, USA) purified using high-performance liquid chromatography with a purity of > 95%, and Complete Freund’s adjuvant (CFA) (Sigma-Aldrich, USA) subcutaneously over six sites at the tail base and on the flank. At 0 and 24 h after immunization, mice were injected intraperitoneally with 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)

Inflammasome activation

Late-log cultures of Salmonella enterica serovar Typhimurium strain SL1344 (a kind gift from Dr. Brad T. Cookson, University of Washington) were grown in L-broth containing 0.3 M NaCl with shaking at 37 °C. Bacteria were washed, resuspended in PBS, and added at a multiplicity of infection of 10 bacteria per macrophage. For activation of NLRP1b, BMM from BALB/c mice were exposed to 1 µg/mL anthrax lethal toxin, prepared as a 1:1 mix of protective antigen and lethal factor (List Biological). …

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

Author did not specify which Lethal Factor (LF) was utilized; List Labs provides the following LF:

• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

… Anthrax Protective Antigen List Biological Laboratories Cat# 171E …

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

Animals and EAE induction procedure

Nine-week-old male C57BL/6J mice aged (n = 14 mice/group) were obtained from Daihan Biolink Co. (Chungbuk, Republic of Korea) for use in this study. …

To induce EAE, mice (n = 14) were immunized subcutaneously on the hind flank with 1 mg/mL myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55; Koma Biotech, Seoul, Republic of Korea) emulsified in complete Freund’s adjuvant (CFA; Sigma-Aldrich, St. Louis, MO, USA) and supplemented with 5 mg/mL Mycobacterium tuberculosis H37Ra (Difco Laboratories, Franklin Lakes, NJ, USA). On days 0 and 2 post-immunization (DPI), 500 ng of pertussis toxin (List Biological Laboratories, Campbell, CA, USA) was injected intraperitoneally into the mice. Control mice (n = 14) remained non-immunized. …

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)