Bacterial Toxin Research Citations

… Anti-PA IgG was measured in rabbit sera via electrochemiluminescence similar to the PA-ECL screening assay. Biotinylated recombinant PA83 (List Biological Laboratories, Inc.) was bound to streptavidin-coated plates (MSD) and used as the capture antigen. …

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

PA Delivery

Anthrax Lethal Factor protease is delivered into cells via co-administration with protective antigen (PA). Cells were treated with 2 µg/mL PA (List Labs, 171E) only as a control, or 2 µg/mL PA + 1 µg/mL Lethal Factor (List Labs, 169L).

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

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

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

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

Pyroptosis inducers and reagents

For activation of the NLRC4 inflammasome, late-log cultures of Salmonella enterica serovar Typhimurium strain SL1344 (a kind gift from Dr. Brad T. Cookson, University of Washington) were grown in Luria-Bertani broth containing 0.3 M NaCl with shaking at 37 °C. Bacteria were washed, resuspended in PBS, and added at a multiplicity of infection of 10 bacteria per macrophage for 90 minutes (unless otherwise indicated). For NLRP1b activation, BMDM from BALB/cJ mice were treated for two hours (unless otherwise indicated) with 1 µg/mL anthrax lethal toxin, prepared as a 1:1 mix of protective antigen and lethal factor (List Biological Labs). For NLRP3 activation, BMDM from C57BL/6 J mice were primed with 100 ng/mL of LPS (List Biological Labs) for three hours, followed by addition of 15 µM nigericin (Cayman Chemicals) for an additional three hours. For pyrin activation, BMDM from C57BL/6 J mice were primed with 500 ng/mL of LPS (List Biological Labs) for three hours, followed by addition of Toxin A (TcdA) from Clostridium difficile (2 µg/mL) (List Biological Labs). For AIM2 activation, BMDM from C57BL/6 J mice were primed with 500 ng/mL of LPS (List Biological Labs), before transfection of 2 µg/mL poly (dA:dT) (Invivogen) at a 1:25 ratio of DNA to lipofectamine (µg/µL) utilizing Lipofectamine 2000 + OptiMEM, as described [53]. Thiomuscimol (Santa Cruz Biotechnology and Cayman Chemicals) and muscimol hydrobromide (Sigma Aldrich) were added at specified concentrations, at the same time as the pyroptosis inducer, unless otherwise specified. The PDI inhibitors PACMA31 and CCF642 (Cayman Chemicals) were reconstituted in 100% DMSO and stored as frozen aliquots at −20°C before use. Each were diluted to target concentrations of 5 µM for CCF642 and 2.25 µM for PACMA31 in cell culture medium, resulting in a final DMSO concentration of 1%.

• 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

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/

• Product #152C – Toxin A from Clostridium difficile

Stimulations and viral infections

N/TERT1 cells were plated at 5 × 104 cells/96-well plate for cytokine release experiments and at 2.5 × 105, 5 × 105, or at 1 × 106 cells per 24-, 12-, or 6-well plate, respectively. The next day, the cells were treated as indicated with 2 µM Val-boroPro (ApexBio and Biozol), 2 µM ANS (Biomol), 100 ng/mL TNFα (PeproTech), 6.5 µM Nigericin (Sigma Aldrich), 0.1 µM B/B homodimerizer (AP20187, MedChem Express), 20 µM Z-VAD-FMK (Peptanova), 10 µM cytarabine (Ara C, Sigma Aldrich), 1 µM MLN4924 (MLN), 1 µg/mL anthrax lethal factor, and 1 µg/mL protective antigen (both List Biological Labs). …

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

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

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

Chemicals and reagents

… LF (List Labs), …

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

… LF (List Labs), …

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