Bacterial Toxin Research Citations

2.3 Immunizations

2.3.1 Intragastric immunizations

Groups of mice were immunized intragastrically on two consecutive days at bi‐weekly intervals with OD 0.5 of formalin‐killed whole cell bacteria corresponding to 1 × 109 bacteria/mL and 7.5 μg of CT (List Biological Laboratories) or 15 μg of mmCT12 with bicarbonate buffer on each occasion (hereafter referred to as IG + CT or IG + mmCT) (Figure 3A).

2.3.2 Sublingual immunizations

Groups of mice were immunized sublingually twice at a bi‐weekly interval with 200 μg of H pylori lysate antigens reconstituted in 7.5 μg of CT or 15 μg of mmCT on each occasion (hereafter referred to as SL + CT or SL + mmCT) (Figure 3A). H pylori vaccination regimen (route and vaccine) was carefully chosen based on our previous studies.14, 18

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Immunizations

To induce local IgA immunity mice were orally immunized with 10 mg of OVA protein grade V (Sigma) dissolved in 0.5 mL of PBS and containing 3% (w/v) NaHCO3 with or without the addition of 10 µg cholera toxin (CT) adjuvant (List Biological Laboratories, Campell, CA) to each dose. The mice were given three oral administrations, 10 days apart, using intragastric intubation. Parenteral immunizations in 0.5 mL PBS were done i.p twice with 50 µg OVA w/wo 2 µg CT.

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Wt-RABV tracing:

For the present trans-synaptic tracing (Figure 1), nine out of eleven cases injected were used from the previous study, in which we focused on cerebellar connections into sensorimotor cortices (Aoki et al., 2019). Before surgery, animals were anesthetized with ketamine (80 mg/kg, Imalgene, France) and xylazine (10 mg/kg, Bayer, Germany). Appropriate levels of anesthesia were monitored by the absence of whisker movements and foot-pinch withdrawal reflex. Additional doses of the ketamine-xylazine mixture were administered i.p. when necessary. After placing animals into a stereotaxic frame (David Kopf Instruments), a mixture (0.15 or 0.2 L) of the French subtype of CVS-11 rabies virus (Wt-RABV, Aoki et al., 2019; Coulon et al., 2011; Raux et al., 1997; Ruigrok et al., 2008; Suzuki et al., 2012; Ugolini, 2010) and cholera-toxin b subunit (CTb, low salt; List Biological Laboratories, 1% w/v in 0.2 M phosphate buffer (PB) at pH 7.4: the injection solution consisted of four parts Wt-RABV and one part CTb) was injected by a 1 l Hamilton syringe in the following functional areas of the cerebral cortex: primary motor cortex (M1, n = 4), secondary motor cortex (M2, n = 4), and medial prefrontal cortex (mPFC, n = 3, one centered on prelimbic and two centered on cingulate cortex). As shown in our previous study, coordinates of the injection sites were determined by reference to the standard rat brain atlas (Aoki et al., 2019; Paxinos and Watson, 2004). The viral stock was kept at 80C until use. This CVS-11 strain of RABV has been confirmed to be transported trans-synaptically in a retrograde direction as well as in the time-dependent manner (Aoki et al., 2019; Kelly and Strick, 2004; Ruigrok et al., 2008; Suzuki et al., 2012; Ugolini, 2010). No neighboring neurons are infected unless they have synaptic contacts to the already infected cells. Adding CTb to the injection solution enabled accurate determination of the injection site (Aoki et al., 2019; Suzuki et al., 2012). Upon the injection, the needle was left in place for another 5 min to allow the virus to spread. After surgery, animals were monitored for signs of stress or discomfort. …

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

… DCs were treated with DMSO (control), CT (1 µg/mL; List Biological Laboratories, Campbell, CA, USA), and CT DMB simultaneously (75 M) for 18 h. …

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… BMDMs were primed with 10 ng/ml ultrapure LPS (List Biologicals, Campbell, CA) for 3 h to induce proIL-1b expression. …

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

… would stimulate cAMP formation in CHO stably transfected with MOR-1B2 or MOR-1C1. In these experiments, PTX (100 ng/ml) (RRID:SCR_014324; List Biological Laboratories, Campbell, CA) which selectively inactivates Gi/Go but does not alter Gs functionality …

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 (List Biological Laboratories, Campbell, CA), …

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

Chondrocyte isolation and culture:

… Cells intended for use in Ca²⁺ analyses were cultured in 96-well plates, and those intended for western blotting and F-/G-actin analyses were cultured in 6-well plates. All cells were cultured for 4 days until near confluence. On day 4, cells were stimulated with LPS (10ng/ml, Escherichia coli 055:B5; List Biological Laboratories, Campbell, CA, USA) or IL-1 (5ng/ml; R&D Systems, Abingdon, UK) or left untreated (controls) for 24h. On day 5, intracellular Ca²⁺ release was measured, cell culture medium was collected, and cells were harvested and immediately frozen and stored at 80C until further analyses.

• Product #203A – LPS from Escherichia coli O55:B5

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Ethics and in vivo experiments:

… EAE was induced in C57BL/6 mice by s.c. immunization with 125µg MOG₃₅₅₅ (MEVGWYRSPFSRVVHLYRNGK) (AnaSpec, Inc.) emulsified in complete Freund’s Adjuvant (CFA) suspension (4mg/ml mycobacteria in IFA), and i.v. injection of 200ng pertussis toxin (List Biological Laboratories) on days 0 and 2 following 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)