Bacterial Toxin Research Citations

Materials

… Pertussis toxin (PTX) was obtained from List Labs (Campbell, CA, USA). …

Induction of EAU in C57BL/6J Mild Model and in B10RIII Severe Model

In this study, 122 C57BL/6J and 41 B10RIII female mice were immunized subcutaneously with 500 µg IRBP(1–20) or 50 µg IRBP(161–180), respectively, emulsified with CFA, and 2.5-mg/mL Mycobacterium tuberculosis H37RA. An intraperitoneal (IP) injection of 1 µg of PTX was administrated to each mouse. …

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)

… mice were injected intraperitoneally with 500 ng boluses of un-nicked diphtheria toxin (DT) (150, List Biologics), every other day. …

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

2.4. Preparation of Toxins

BoNT/A was harvested from C. botulinum strain ATCC19397 following a 6-day anaerobic culture as described previously [18,19]. BoNT/B complex was purchased from List Bio Laboratory Inc. (Campbell, CA, USA). The potencies of the toxins were determined using standard mouse bioassays with established LD50 values for BoNT/A and B. The LD50 values of BoNT/A and B in mice were confirmed to be 0.1 ng and 0.18 ng, respectively.

Luminex multiplex binding assay

The specificities and avidities of clonal IgGs in culture supernatants were determined in a multiplex bead assay (Luminex Corp.) as described (25, 44) with modifications. Culture supernatants were diluted in Luminex assay buffer (phosphate-buffered saline [PBS] containing 1% bovine serum albumin [BSA], 0.05% NaN3, and 0.05% Tween 20) with 1% milk and incubated for 2_h with the mixture of antigen-coupled microsphere beads in 96-well filter-bottom plates (Millipore). After washing three times with assay buffer, the beads were incubated for 1_h with PE-conjugated mouse anti-human IgG Abs (JDC-10; Southern Biotech). After three washes, the beads were resuspended in assay buffer and the plates read on a Bio-Plex 3D suspension array system (Bio-Rad). Antigens and controls included BSA, mouse anti-human Ig(κ) (SB81a; Southern Biotech), mouse anti-human Ig(λ) (JDC-12; Southern Biotech), mouse anti-human IgG (Jackson ImmunoResearch), tetanus toxoid from Clostridium tetani (List Biological Laboratories), …

• Product #191 – Tetanus Toxoid from Clostridium tetani

NLRC4 Activity in Human Monocytes

A 10-µL aliquot of compound (10X in 1% DMSO) was added to PMA-differentiated THP1-Null2 cells (65,000 cells in 80 µL of RPMI media per well) and incubated for 60 minutes in a humidified incubator at 37°C with 5% CO2. NLRC4 activation was initiated by adding a 10 µL mixture of LFnNeedle and B. anthracis protective antigen (PA) (List Labs #171E) (10X). LFn-Needle is fused to the amino-terminal domain of LFn (in house). This fusion system, when co-administered with the anthrax toxin’s PA, allows intracellular delivery of the bacterial ligand [11]. OptiMEM (10 µL) was added to control wells in lieu of LFnNeedle/PA treatment. The plate was incubated for 5 hours in a humidified incubator at 37°C with 5% CO2.

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

Reagents

… Pertussis toxin (181) was purchased from List Biological. …

Induction of active EAE

In 6- to 8-week-old mice, EAE was induced by s.c. injection of myelin oligodendrocyte glycoprotein 35–55 peptide (100 µg/mouse) emulsified in complete Freund’s adjuvant including heat-killed Mycobacterium tuberculosis (200 µg/mouse) on day 0, along with intraperitoneal (i.p.) injection of pertussis toxin (200 µg/mouse) on days 0 and 2. …

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

… cholera toxin (List Biological, Campbell, CA) for 10 days. …

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

2.13. Priming of Inflammasomes

Inflammasome activation was measured according to Primiano et al.’s description [28]. HCM3 cells were plated in EMEM without supplements at 1 × 105 cells/well. Cells were stimulated for 1 h with 10 g/mL (13.4 mM) of nigericin (tlrl-nig, InvivoGen, San Diego, CA, USA) for 2 h with 2 mM ATP (tlrl-atpl, InvivoGen), or overnight with 200 g/mL of monosodium urate crystals (MSU; tlrl-msu, InvivoGen) for NLRP3 induction; flagellin/DOTAP complex, obtained with 25 μL HBS buffer with 500 ng flagellin (final concentration at 1 μg/mL; tlrl-stfla, InvivoGen) and 3 μL DOTAP (11202375001, Sigma-Aldrich) for each well for NLRC4 induction; with 0.5 mL Opti-MEM supplemented with GlutaMax (Opti-MEM™ Reduced Serum Medium containing GlutaMAX™ Supplement, 51985034, Gibco, Grand Island, NY, USA) containing 1 μg/mL Bacillus anthracis lethal factor (172B, List Biological Laboratories, Campbell, CA, USA) for 3–6 h for NLRP1 induction; and with a poly(dA:dT)/lipofectamine complex, obtained by mixing 50 μL Opti-MEM containing 1 μg poly(dA:dT) (final concentration at 2 μg/mL) with 50 μL Opti-MEM containing 2.5 μL lipofectamine 2000 (11668027, Thermo Fisher’s Scientific, Waltham, MA, USA) for each well for AIM2 induction. …

• Product #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis

… with 20 mg of D-galactosamine (DGalN, MilliporeSigma), to sensitize them to the biological effects of endotoxin, and simultaneously challenged with 5 ng of E. coli O111:B4 LOS (List Biological Laboratories, Campbell, CA) [23]. …

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

Biodistribution study of nanoparticles in EAE mouse model

The biodistribution of nanoparticles was assessed in the EAE mouse model. Female C57BL/6 mice (11 weeks old) were subcutaneously injected with 200 μg of MOG35-55 peptide (2 mg/mL, Prospec, Rehovot, Israel, cat# PRO-371), emulsified with an equal volume of Complete-Freund’s adjuvant (CFA) (Chondrex, Woodinville, WA, USA, cat# 7001), consisting of 4 mg/mL of heat-killed M. Tuberculosis H37 RA. Subsequently, the mice received 400 ng of Pertussis toxin (PTX) (List Biological Labs, Campbell, CA, USA, cat# 180) intraperitoneally at 2 and 24 h post-injection of the emulsion. Intravenous injection of Cy5-labeled nanoparticles at a dose of 40 mg per kg was performed on day 12 post-immunization. The fluorescence of nanoparticles in the brain, spinal cord, and major organs was assessed using an IVIS spectrum In Vivo Imaging System (PerkinElmer) at various time points post-injection. …

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)