Bacterial Toxin Research Citations

IN VIVO EXPERIMENTAL HUMAN ENDOTOXEMIA

Human endotoxemia experiments were conducted at the research unit of the Intensive
Care department of the Radboud university medical center according to our standard
protocol (21). Subjects refrained from caffeine and alcohol in the 24 h before the
experiment and from food and drinks 10 h before the experiment. One venous canulae
was placed in the antecubital vein, for fluid infusion and endotoxin administration.
Subjects were pre-hydrated in the hour before LPS administration (1.5 L 2.5%
glucose/0.45% saline) followed by 150 mL/h for 6 h (until the end of the experiment).
Blood pressure monitoring and blood withdrawals were performed using an arterial
cannula placed in the radial artery. In addition, ECG was continuously monitored and
vital signs were recorded every 5 seconds on a Phillips MP50 patient monitor using an
in-house developed data capturing system. Body temperature was measured every 30
mins with infrared tympanic measurements (FirstTemp Genius 2, Covidien Geldermalsen,
the Netherlands). At T=0, 1 ng/kg bodyweight LPS (E. coli type O113, Lot no. 94332B1;
List Biological Laboratories, Campbell, USA) was administered intravenously to elicit a
transient systemic inflammatory response.

• Product #9433 – GMP LPS Lipopolysaccharide List™ Hpt™ From Escherichia Coli Type O113

… Pertussis toxin List Biological Laboratories, Hornby, ONT, Kanada; Cat # 181 …

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

… To facilitate lymphocyte transfer to the CNS, 300 ng of pertussis toxin (List Biologicals, Campbell, USA) was administered intraperitoneally two hours and two days after EAE induction (Contarini et al., 2018). …

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)

… rPA83 B. anthracis WB (40 ng) c = 1 mg/ml; List Biologicals, Campbell, USA …

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

… DT powder is purchased from Sigma-Aldrich #D0564 or List Biological Labs #150 and can be stored at 2-8 °C …

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

Cell culture and treatment with S100A4

BMDCs were generated by culturing bone marrow cells in culture medium (RPMI-1640; Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco), 1% L-glutamine, 1% gentamicin, and 50 μM 2-mercaptoethanol (all from Sigma-Aldrich) in the presence of 200 ng/ml recombinant Flt3L (PeproTech) for nine days. Cells were incubated in vitro with various stimuli, including CT (List Biological Laboratories) (0.1 μg/ml), …

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

Stereotaxic injections and retrograde tracing

Mice used in the experiments were Skor2GFP/wt (n=6). Mice were anesthetized with isoflurane, attached to the stereotaxic frame, and a small hole was drilled into their skull. For retrograde tracing of vlPAG neurons, bilateral, intracranial injections of 0, 2% Cholera toxin B subunit (#104; List Biological Lab.Inc.) were injected at the speed of 50 nl/min using a microinjector (UltraMicroPump III, World Precision Instruments) and microsyringe (Hamilton 7803-06). The stereotaxic coordinates for injections were (measured from bregma, in mm): -5,19 to -5,4 (AP); 0,88 (ML); -4,4 (DV). The coordinates were obtained from the mouse brain atlas (Paxinos and Franklin, 2012). Mice were intracardially perfused 5-7 days after the injections and the brains were collected. Brains were processed for vibratome sectioning and further for IHC stainings and analysis for GFP and CtB expression in the midbrain.

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

Flow cytometry

… Cryopreserved liver or PBMCs were thawed and rested overnight in sterile R10 media (RPMI 1640, Corning), supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA), penicillin/streptomycin, and L-glutamine plus 10 U/mL DNAse I (Roche Life Sciences, Indianapolis, IN) at 37°C, 5% CO2, and 95% humidity incubation conditions. Cells were stimulated as previously described,45 with a final concentration of 2 mg/mL AAV8 peptide pool C, hOTC peptide pool A or 1 μg/mL Staphylococcal enterotoxin B (SEB) (List Biological Laboratories, Campbell, CA) as a positive control. …

… Incomplete Freund (DF0639-60-6) and Mycobacterium tuberculosis (DF3114-33-8) from Difco (Detroit, MI, USA). Pertussis toxin was obtained from List Biological Lab. (#181)…

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

STAR★Methods
Key resources table

Diphtheria toxin (mutant CRM197) List Biological Laboratories Cat. no. 149A

Enzyme linked immunosorbent assay (ELISA)

An ELISA to determine the development of antibodies against diphtheria toxin (DT) after long time DT treatment was performed according to Buch et al. (Buch et al., 2005). For the assay, a 96-well plate was coated with 2μg/mL DT (mutant CRM197, List Biological Laboratories) at 4°C overnight and the free binding sites of the plate were blocked the following day with 0.5% BSA in PBS for two hours at RT. …

• Product #149A – Diphtheria Toxin CRM197, Mutant