Bacterial Toxin Research Citations

Mucosal sensitisation model

The animal study protocol was approved by the “Service Vétérinaire du Canton de Vaud”, Switzerland and the experiments were performed at Ambiotis (Toulouse, France). The model has been previously described in detail (Zuercher et al., Citation2012). Briefly, five-week-old conventional female BALB/cOlaHsd mice from Envigo (Gannat, France) were sensitised for a total of seven consecutive weeks by weekly gavage of 50 mg ovalbumin (OVA, Merck) [sensitised group (S.)] or water [non-sensitised group (N.S.)] in combination with 20 μg cholera toxin (CT) (List Biologicals, purchased from LuBioscience, Lucerne, Switzerland). On day 67 or 68, mice were challenged with an oral administration of 200 mg ovalbumin (OVA; Sigma, Switzerland) and harvested at day 68 or 69 (Supplementary Figure 1A). Two independent experiments were performed with n = 8 animals per group for the exploratory experiment and n = 13 animals per group for the confirmatory experiment.

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

Drugs and Pharmacology

Formalin (2%, Sigma), PEG (30%, average M.W. 1500, Acros Organics), CFA (50%, Sigma), LiCl (12.5 mg/mL, Sigma), CNO (0.1 mg/mL, Tocris) and Diphtheria toxin (DT, 0.5mg/ml, List Labs) were diluted in normal saline. … DT was injected intramuscularly at a volume of 5 µL/g body weight. Mice received 3 DT injections under light isoflurane anesthesia with 48 h between injections.

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

… Goat anti-CTb (1:5000, IHC) List Biological Labs 703 …

CTb injections

Hindpaw: Mice were anesthetized (5% induction, 2-3% maintenance). Injections of 1% unconjugated CTb
(~7µL across four sites) were injected into the ventral paw of C57BL/6 wild type mice to label paw sensory
input. Animals were perfused 5 days after CTb injection and tissue was processed for IHC as described above.
Free floating sections were immunostained with goat-CTb (list-labs). 5x images of CTb-labeled afferent inputs
were taken at the Zeiss LSM 800 confocal microscope and ImageJ was used for synaptic analysis.

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

… Rorctd Tomato DTR mice were injected with diphtheria toxin (10 ng/g, i.p.; List Biological Laboratories, Campbell, CA, USA) using single or continuous injections, as indicated …

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

… The fluorescence intensity was analysed using FlexStation 2 (Molecular Devices, Ex/Em = 490/525nm). Where appropriate, cells were treated with PTX (100ng/ml; List Biological Laboratories) for 16?h and with YM-254890 (1µM; a gift from Dr. J. Takasaki60) for 45min before the assay. …

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)

Toll-like receptor (TLR) 4 and 5 activity assays

TLR4 and TLR5 activity assays were performed as described previously with minor modifications (14, 15). Briefly, HEK-Blue-hTLR4 cells and HEK-Blue-hTLR5 cells carrying a secreted embryonic alkaline phosphatase (SEAP) reporter construct were obtained from InvivoGen (CA). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 0.5% penicillin/streptomycin with and without 0.2% Normocin (InvivoGen, CA) for TLR5 cells and TLR4 cells at 37°C with 5% CO2, respectively. Monolayers of 1 × 105 cells per well in a 96-well plate were incubated with media alone, PBS, and FlaA and FlaB proteins at different concentrations ranging from 10 pg/mL to 10 µg/mL for 24 h. E. coli lipopolysaccharide O111:B4 (List Biological Laboratories, Inc., CA) was added to HEK-Blue-hTLR4 cell as a positive control. …

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

Tdap-specific IgG and IgG subclass detection

All available leftover maternal and cord serum samples from both deliveries were tested at the Université Libre de Bruxelles (ULB). Total and subclass Tdap-specific IgG antibodies were quantified using an in-house bead-based multiplex immunoassay (28). In brief, serum samples were incubated with distinct fluorescent magnetic beads, individually coated with specific purified target antigens: PT (List Labs, #180), FHA (Sigma, #F5551), PRN (NAC, #NAT41666), diphtheria toxin (DT, List Labs, #151) and tetanus toxin (TT, MassBiologics, # LP1105P). Samples were measured at appropriate dilutions with a Multigam reference (CAF-DCF, Belgium), a WHO international standard NIBSC 06/140 and blanks included on each plate. Serum dilutions used to titrate IgG and IgG1 were 1:9000-1:18000-1:36000, 1:70-1:140-1:280 for IgG2 and IgG3, and 1:70-1:700-1:7000 for IgG4. After washing, the captured IgG was detected with R-phycoerythrin labelled antibodies specific for each isotype (IgG1-4). …

• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #151 – Diphtheria Toxoid, from Corynebacterium diphtheriae

Pertussis toxin List Biological Laboratories, Campbell, CA Cat# 181238A1

EAE induction

EAE was induced in female C57BL/6 mice (8–10 weeks of age).38 Mice were subcutaneously immunized with 200 mg of the MOG35–55
peptide in a 100-ml solution (containing 50% complete Freund’s adjuvant with 10 mg/ml of heat-killed M. tuberculosis H37Ra strain).
The MOG35–55 peptide was injected into areas that drained into the axillary and inguinal lymph nodes. Pertussis toxin (200 ng/mouse) was administered intraperitoneally on the day of immunization and again at 48 h later. Animals were examined for disease symptoms
daily. The evaluation of disease severityEAE induction. …

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

Induction of EAE

EAE was induced in male C57BL/6 J mice (Jackson Laboratories) at 8–12 weeks of age by subcutaneous immunization with an emulsion containing 200 μg of myelin oligodendrocyte glycoprotein (35–55) (MOG35–55) in saline and an equal volume of complete Freund’s adjuvant (CFA) containing 400 ng of mycobacterium tuberculosis H37RA (Difco Laboratories, Detroit, MI). All mice were administered 400 ng of pertussis toxin (List Biological, Campbell, CA) intraperitoneally (i.p.) at 0 and 48 h post-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)

4.2. Air Pouch Model

To evaluate the possible immunomodulatory effects of PHA 568487 in vivo, the air pouch model of acute inflammation was used [12,24]. At 8 weeks of age, male C57BL/6JRj mice (Janvier Labs, Le Genest-Saint-Isle, France) were injected with 3 mL of sterile air between the scapulae under
isoflurane anaesthesia. Three days later, the air pouch was re-filled with 3 mL of sterile air. On the sixth day, the mice received an intraperitoneal injection of either PBS (Thermo Fisher Scientific, Waltham, MA, USA) or PHA 568487 (Tocris Bioscience, Ellisville, MO, USA) at 12.4 μmol kg-1 (5 mg/kg) or 123.4 μmol kg-1 (50 mg/kg). 15 min later, all mice were injected with LPS (10 μg, List Biological Laboratories Inc., Campbell, USA) directly into the pouch. The mice were sacrificed six hours after the LPS-injection by an overdose of pentobarbital (Apoteket AB, Stockholm, Sweden). …

Author did not specify which List Labs LPS product was utilized in their research.
List Labs provides the following LPS products: https://www.listlabs.com/product-information/lipopolysaccharides/