Bacterial Toxin Research Citations

… B. anthracis LF was purchased from List Biological Laboratories. …

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

Reagents:

… B. anthracis lethal factor (LF) was acquired from List Biologicals (#172C). …

J774.A1 culture and stimulation:

J774.A1 cells were cultured in IMDM (Lonza) containing 10% (vol/vol) heat-inactivated FBS, 1% (vol/vol) nonessential amino acids (Lonza), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37C in a humidified atmosphere containing 5% CO₂. Cells were scraped for passages and, for experiments, counted and seeded into 96-well plates in culturing medium. On the next day, cells were changed to fresh media and stimulated with vehicle or 1 g/ml PA combined with 0.5 g/ml LF (LeTx), VBP (0.1% TFA in DMSO) or 1G244 (DMSO) at a final concentration of 10 M or 25 M.

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

EXPERIMENTAL –  FRET Lethal Factor Protease Assay:

The FRET assay measures the enzymatic activity of LF with an internally quenched peptide substrate derived from MAPKK. LF cleavage of the substrate separates the quencher and fluorescent tag leading to an increase in fluorescence intensity. The slope of fluorescence absorbance is therefore used to determine enzyme activity. Anthrax lethal factor (30 nM final concentration) and MAPKKide substrate (25 M final concentration) were purchased from List Biological Laboratories, Campbell, CA. …

Author did not specify which MAPKKide substrate was utilized; List Labs provides the following:

• Product #530 – MAPKKide Peptide Substrate (o-Abz/Dnp) for Anthrax Lethal Factor
• Product #531 – MAPKKide Peptide Substrate (DABCYL/FITC) for Anthrax Lethal Factor
• Product #532 – MAPKKide Plus (AMC) Specific Substrate for Anthrax Lethal Factor

Assessment of toxin neutralizing antibodies

Toxin neutralization assay was performed as previously described^4,5,6. Briefly, sample dilutions were added to J774 macrophages cultured in cultured in RPMI supplemented with 10% fetal calf serum. Bacillus anthracis lethal toxin (LeTx) [i.e., PA plus Bacillus anthracis lethal factor (LF, List Biological, Campbell, CA)] was then added to the plates. After overnight incubation, MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide; Sigma-Aldrich) was added to assess the viability of macrophages as a function of redox potential. Toxin neutralizing antibody titers were determined as the lowest concentrations of sera  that protect macrophages from the cytotoxicity of LeTx.

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

Antibodies:

Primary antibodies used in this study that are commercially available include: rabbit anti-MEK2 (Santa Cruz Biotechnology sc-523, AB_2281672), goat antiprotective antigen from Bacillus anthracis(List Biological Laboratories #771B), …

LFNEffector intoxication of T84 cells:

… For intoxication of cells, media was replaced over 105 cells previously seeded in a 12-well plate. 31.7 nM PA alone (List Labs, #171E) or in the presence of 13.6nM LFN, LFN-ACD, or LFN-RID was added to media and cells were incubated for 20 hr at 37C in the presence of 5% CO2. For co-intoxication of 13.6 nM LFNACD and LFN-RID, 3x excess PA was used to ensure equal translocation of both effectors.

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

… Recombinant anthrax lethal factor was purchased from List Biological Laboratories, Inc. …

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

Test approach:

No standardized approaches are available to investigate anthrax seroprevalence in wildlife. Thus, samples were tested for antibodies against the anthrax protective antigen (PA, List Biological Laboratories Inc) using an in-house ELISA and an in-house Western Blot and for antibodies against anthrax lethal factor (LF, List Biological Laboratories Inc) using an in-house Western Blot. …

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169L (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

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

… QD grade anthrax lethal factor (LF-A 0.1 mg, product No. 169 A, B), (List Biological Laboratories. Inc.).

List Labs Product #169A/B (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence; Lyophilized) has been discontinued; however, Product #169L (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence; Liquid) is available for purchase.

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

Intestinal organoid culture and live imaging:

Primary intestinal epithelial organoids were grown as described before⁵⁶. Briefly, the small intestine and colon were flushed and cut into small pieces that were dissociated in PBS containing 2mM EDTA for 30min at 4C. After extensive washing, the isolated crypts were pelleted and mixed with 25l of Matrigel (Corning) and put in a 24-well plate. After polymerization of the Matrigel, complete culture medium containing advanced DMEM/F12 (Gibco) supplemented with B27 supplement (0.02%, Invitrogen), N2 supplement (0.1%, Invitrogen), N-acetylcysteine (0.0025%, Sigma-Aldrich), mouse epidermal growth factor (mEGF; 0.001%, Invitrogen), and conditioned Rspondin and mNoggin medium was added to the wells. Organoids were seeded and imaged in an 8-well chamber (iBidi) for cell death analysis by real time lapse microscopy or in 24-well plates for Western blotting analysis. Cell death was induced with TcdA (1g/ml, Enzo Life Sciences), TcdB (1g/ml, List Laboratories), …

• Product #155 – Toxin B from Clostridium difficile