Bacterial Toxin Research Citations

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.

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)

3.2. Reagents

We obtained purified lipopolysaccharides (LPS) from Escherichia coli from List Bio-logical Laboratory (Campbell, CA, USA), which we dissolved in endotoxin-free water. …

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/

Chemical reagents

… Cholera toxins from Vibrio cholerae (product no.100B: Lot no. 10071A2) were purchased from List Biological Laboratories (Campbell, CA, USA). …

Human colonoid swelling assays

Human epithelial crypt-like cells were obtained from colon tissue biopsies. Crypt isolation was performed as previously described with minor modifications [23]. … Swelling of the human colonoids was monitored by random imaging every 5 min using the BioTek Cytation™ 5 Cell Imaging Multi-Mode Reader after treatment with forskolin (5 μM), forskolin plus nornidulin, cholera toxin (2 μg/mL), or cholera toxin plus nornidulin. Swelling of colonoids was calculated as the relative area increased from 0 min to 60/240 min and analyzed as the area under the curve (AUC) using CellProfiler 4.2.1 program.

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

Anthrax lethal toxin neutralization assay

RAW264.7 cells were seeded as mentioned above and cultured in DMEM supplemented with 10% FBS and 10 mM HEPES (Fisher Scientific, Fai Lawn, NJ, USA) overnight. The toxin neutralization assay was performed as previously described [73]. Pooled serum samples from vaccinated guinea pigs before challenging with Bcbva UFBc0009 and B. anthracis Ames UF00738 spores were prepared at 200-, 1,200-, and 12,000-fold dilutions in DMEM supplemented with 10% FBS and 10 mM HEPES. Pooled serum samples from unvaccinated animals served as a control. The diluted serum samples were then incubated with 50 ng/ml PA (List Labs, Campbell, CA, USA) and 160 ng/ml LF (List Labs, Campbell, CA, USA) for 30 min before adding to RAW264.7 cells. …

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

Author did not specify which Lethal Factor (LF) was utilized; List Labs provides the following LF:

• Product #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

Mouse treatments. Mice received 3 doses of i.p. tamoxifen (Sigma Pharmaceuticals) over one week. Tamoxifen was suspended in corn oil and delivered at a concentration of 0.2 mg/g per dose. Animals were rested for 4 weeks after the final tamoxifen dose (13, 38). E. coli LPS O55:B5 (List Biological Laboratories) was instilled into the tracheas of mice sedated with isoflurane (Baxter) at a concentration of 20 μg in 50 μL of PBS using a modified gel-loading tip with direct laryngoscopy (13). Bleomycin (TEVA Pharmaceuticals) was administered at a dose of 1.5 U/kg in 50 μL of PBS by the same method (21). Mice were euthanized by i.p. injection of 10% Fatal Plus solution (Vortech Pharmaceuticals). Three to 4 mice were treated per time point in each inflammatory model.

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

… Eight days after oral administration of OVA, 1 mg OVA + 20 µg cholera toxin (100B; List Biological) in 0.2 M sodium bicarbonate were provided by gavage once per week …

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

Animals

… Male, Advillin-Cre+/−, mice were crossed with female, CalcaWT/GFP (Figure 1D), mice to generate CalcaWT/GFP, CalcaWT/DTR (Figure 1C), or wildtype CalcaWT/WT (Figure 1A) offspring. Diphtheria toxin (DTX; List Biological Laboratories, Product #150) was reconstituted to 20 μg/mL in sterile 0.9% saline, aliquoted, and stored at −80°C until used for injections. Once animals reached 10–12 months of age, DTX was administered by i.p. injection (100 μg/kg) twice with 72 h between the two injections. Animals were sacrificed and prostate tissue was harvested 14 days after the second injection (Figure 1E).

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