Bacterial Toxin Research Citations

Animals and immunization

Female inbred C57BL/6, SJL/J, (C57BL/6xSJL/J)F1, and DBA/1J mice were purchased from The Jackson Laboratory. B6.129(Cg)Foxp3tm3(DTR/GFP)Ayr/J (FOXP3DTR) breeder pairs were purchased from The Jackson Laboratory and were bred in house at the University of Florida. Animals were approximately 8–10 weeks old at the time of disease induction. EAE was induced by 2 subcutaneous injections each containing 100 μg peptide (MOG35–55, MOG119–132, prevention of MOG79–96, MOG92–106, and MOG1–125) or 50 μg peptide (MOG119–132/MOG35–55 for a mixed total of 100 μg, and reversal of MOG79–96) emulsified in CFA (Sigma Aldrich) supplemented with 9 mg/mL Mycobacterium tuberculosis (Difco Laboratories). Pertussis Toxin (200 ng or 500 ng for MOG92–106 induction only, List Biological Laboratories, Inc., was injected intraperitoneally 2 and 24 h after immunization with CFA. All peptides were based on mouse proteins.

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)

… Cholera toxin (CT; Cat#10654, Lot#10067A1) was obtained from List Biological Laboratories, Inc. (Campbell, CA, USA). …

2.9 Mouse closed-loop models of cholera toxin (CT)-induced diarrheas

ICR mice (30–35 g) were purchased from Nomura Siam International Co., Ltd., Thailand. Mice were acclimated under controlled conditions for 3 days (temperature 22 ± 1°C; relative humidity 30–70%; 12-h dark/light cycle) with access to food and water ad libitum. To investigate the in vivo efficacy of DHLV on CT-induced intestinal fluid secretion, mice were fasted for 24 h before experiments. Mice were anesthetized with an intraperitoneal injection of thiopental (50 mg/kg), abdominal incision was made, and ileal closed-loops (length of 2–3 cm) were created by ligation. For intestinal fluid secretion, ileal closed-loops were instilled with 100 μl of sterile PBS containing CT (1 μg/loop) with or without intraluminal (i.l.) or intraperitoneal (i.p.) administration of DHLV (20 μM and 2 mg/kg, respectively). The abdominal incision was closed by sutures and mice were allowed to recover from anesthesia. Six hours later, mice were euthanized and ileal closed-loops were collected. Weight/length ratios of ileal closed-loops, which indicated intestinal fluid secretion, were measured. For baseline intestinal fluid transport, ileal closed-loops were instilled with 200 μl of PBS with or without DHLV (20 μM). One and thirty min later, mice were euthanized by intraperitoneal injection of overdose of thiopental (150 mg/kg) and ileal closed-loops were collected and measured for weight/length ratios. Mice were euthanized with an injection of thiopental (150 mg/kg).

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

Materials and reagents were obtained from … LF, PA83, and PA63 were obtained from List Biological Laboratories (Campbell, CA), …

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #174 – Anthrax Protective Antigen, Activated (PA 63) 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

E2 pellet implantation and induction of EAE:

… Mice were then immunized with 200µg mouse MOG-35-55 peptide (PolyPeptide Laboratories, San Diego, CA) in 200µg Complete Freund’s adjuvant [Incomplete Freund’s adjuvant (IFA, Sigma-Aldrich, St. Louis, MO) with heat-killed Mycobacterium
tuberculosis (Mtb, Difco, Detroit, MI)] subcutaneously along the flanks at four sites. Additionally, mice
were administered 75ng of pertussis toxin (Ptx, List Biologicals, Campbell, CA) via an intraperitoneal (i.p.) injection on the day of immunization and 200ng i.p. 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)

EAE induction and score

… EAE induction was performed by mixing rhMOG (200 mg/mouse) in CFA (Difco Laboratories, DF3114-33-8) containing Mycobacterium tuberculosis H37Ra (Difco Laboratories, DF0639-60-6) and injecting s.c. into the abdomen bilaterally (50 ml emulsion in each side). Pertussis toxin (120 ng) (List Biological Laboratories, 180) diluted in PBS was administered i.p. on days 0 and 2 after immunization. Control mice receiving PBS in CFA were received injections
with the same amount of pertussis toxin. …

• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer

… To induce hair cell damage, DT (List Biological Laboratories, Inc., Campbell, CA) at 25 or 50 ng/g was injected intramuscularly at 7 weeks of age (1-week post-tamoxifen) once a day on two days spaced 44-48 hours apart. We selected a DT dose for each experiment based on periodic tests of DT potency, which varied with each lot and diminished over time during storage at -20°C …

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

2.2. EAE induction and monitoring

EAE mice were induced by subcutaneous immunization with 50 μg of MOG35–55 (MOG001, Chinese Peptide, China) emulsified in CFA (7001, chondrex, U.S.A., 1.5 mg/ml). Intraperitoneal injection of 300 ng Pertussis toxin (181, List Biological Laboratories, U.S.A.) was administered at the time of induction and again 48 h later. Control mice were treated with CFA and Pertussis toxin alone. …

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

… Pertussis toxin List Biological Cat# 180 …

EAE induction

… For passive transfer induced EAE, naive CD4 T cells were differentiated under Th17-skewing conditions for 96 h, then IL-17A-GFP positive cells were sorted by using a FACSAria (FACSAria III, BD) and injected intravenously into Rag1−/− mice (1 x 106 cells per mouse). Two days after T cell transfer, recipient mice were immunized with MOG35–55 peptides (300 μg/mouse) emulsified in complete Freund’s adjuvant, containing 5 mg/mL heat-killed Mycobacterium tuberculosis H37Ra, and 200 ng pertussis toxin (PTX, dissolved in 0.1 mL of PBS) was injected intravenously. Mice were then observed daily for disease development. …

• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer

Immuno-fluorescent visualization of the triple labeling in the INC/DN.

The 30-μm coronal sections were incubated with goat anti-CTB polyclonal antibody (1:200; List Biological Labs, Campbell, CA, USA) and the rabbit anti-PHA-L overnight at 4–8°C. Next day, the sections were incubated in the cocktail of the same conjugated 568 and 488, plus donkey anti-goat conjugated either Alexa Fluor 350 or ByLight 405 to reveal CTB labeled pre-oculomotor neurons; however, the Alexa Fluor 350 was for conventional fluorescent microscopy and the ByLight 405 was for confocal microscopy observations.

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

2.3.9. Immunohistochemistry for VGlut1

Following ex vivo MRI, spinal cords were washed in PBS, cryoprotected in 30% sucrose, frozen in OCT compound and cut in transverse 15 μm sections. Sections were immunostained using; rabbit anti-vGlut1 antibody (1:1000, #135302, Synaptic Systems), Goat-anti-CTB (1:2000, #703, List Biolgicals), Mouse-anti-NeuN (1:500, #MAB377, Millipore). Sections were counterstained with Alexa 546 and Alexa 488 (1:1000, #A10040 and #A10036, Thermofisher) and Dylight 650 (1:500, #ab96938, Abcam). Five sections throughout the L4-L6 segments were selected and traced α-motor neurons (Ctb+/ NeuN+) in the ventral horns were imaged at 400× magnification (LSM 710, Zeiss). Animals with fewer than five traced motor neurons were excluded from analysis.

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