Bacterial Toxin Research Citations

Cell culture:

The human monocytic leukemia cell line THP-1 (Cell Lines Service GmbH) was cultured in RPMI supplemented with 10% fetal calf serum (FCS, Life Technologies, Basel, Switzerland) and used below passage 10. THP-1 cells were differentiated into macrophages in the presence of 50 ng/ml phorbol myristate acetate (PMA, Sigma, USA) for 48 hours. THP-1 PMA-derived macrophages were stimulated with 10 ng/ml E. coli LPS (List Biological Laboratories, California) for 2 hours. …

Author did not specify which List Labs E. coli LPS product was utilized in their research.  List Labs provides the following LPS products:  https://listlabs.com/product-information/lipopolysaccharides/

Materials and Methods

Anthrax lethal factor (Product #172 or #169), are products of List Biological Laboratories, Inc. The 96-well, black, flat bottom, non binding plates used for the fluorescent plate assay were from Corning (cat # 3991). Bovine plasma (cat # 7310806) was
purchased from Lampire Biological Laboratories.

Sample Preparation: Stock solutions of the fluorogenic substrates, MAPKKide® Plus (± biotin), were made 2.5 mM in
DMSO based on the peptide content determined by elemental analysis. The substrates were diluted in assay buffer: 20 mM
HEPES, pH 8.0 containing 0.1% Tween-20.

LF Activity Assays:
A rapid microplate assay method was evaluated for two ranges of LF: 10 to 1000 pg LF/ml 1:10 diluted bovine plasma and 5 to 250 ng/ml 1:5 diluted bovine plasma. Dilution of the bovine plasma was necessary in order to minimize background.
For the method detecting higher levels of LF (5 to 250 ng/ml 1:5 diluted bovine plasma), 10 μM MAPKKide Plus was added directly to the 1:5 diluted bovine plasma, and the assays were run at 37°C using the kinetic mode of the plate reader with
readings at 1 or 3 minute intervals. The samples were run in triplicate with 9 replicate blanks. The limit of detection was
calculated from the normal distribution of the blank samples (mean + 3 stdev; n = 9).
For the range 10 and 1000 pg/ml 1:10 diluted plasma, 1.25 μM MAPKKide Plus was added directly to the diluted bovine
plasma and the time-dependent increase in fluorescence was monitored at 37°C hourly for 5 hours. For the blank samples
containing no LF there were 3 sets of quadruplicates and the standard deviation was calculated from these three sets. At
each time point, the plate was read 5 times to increase the precision of the fluorescence readings. The standard curve was
analyzed using a linear regression fit forcing the intercept through the mean value of the blanks. The limit of detection was
calculated from the normal distribution of the blank samples (mean + 3 stdev; n = 3 sets of quadruplicates) and calculated as
pg LF/ml plasma using the standard curve. Subsequently, 6 data sets were evaluated, 2 data sets per day for 3 consecutive
days. The data is presented as the average of these 6 data sets, each with 4 replicate samples and 12 replicate blanks. At
each time point, the plate was read 3 times to increase the precision of the fluorescence readings.

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #532 – MAPKKide® Plus (AMC) Specific Substrate for Anthrax Lethal Factor

… A total of 200 ng of Pertussis Toxin (List Biological Laboratories) was given ip on day 0 and 2. …

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)

… Samples were challenged with 10 ng mL-1 of lipopolysaccharide (LPS, List Biological Laboratories inc.) and treated with PBS or increasing concentrations of AZ6983, 0.1, 1, 10 or 100 mol L-1. …

Author did not specify which List Labs LPS product was utilized in their research.  List Labs provides the following LPS products:  https://listlabs.com/product-information/lipopolysaccharides/

Mouse immunization:

Fresh leaves were crushed while frozen and mixed in distilled water. The quantity of leaf matter administered to each mouse was adjusted to deliver 0.2 µg of AO, and it was mixed with or without CTB (List Biological Laboratories, U.S.A.) (1 µg per mouse). …

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

… and the retrograde tracer cholera toxin subunit b (CTb; low salt, 1% in distilled water, List Biological Laboratories Inc., Campell, CA) were used …

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

Subjects:

Two male macaque monkeys (1 Macaca mulatta (Y), 1 Macaca nemestrina (S)) were used for these experiments. Animal Y was 4 years old and weighed 8.2 kg and Animal S was 6 years old and weighed 17.4 kg at the time of surgery. The subjects used in this study were anatomically typical, with no observable abnormalities. MRI-guided injections of anatomical tracers were performed stereotaxically under sterile conditions. Injection sites, tracers used, and volumes of injection for each case are shown in Table 2.1. The animals received bilateral injections of anterograde tracer (fluoroemerald; FE) in superior colliculus and retrograde tracer (choleratoxin B; CTB) in the lateral nucleus of the amygdala; this yielded 4 hemispheres for anatomical analysis.

Table 2.2

Choleratoxin B List Labs

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

… lipopolysaccharide (LPS; Escherichia coli-derived, serotype O111:B4; List Biological Laboratories, Campbell, CA); …

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

… and S1P 1 LysM KO mice immunized with an emulsion containing 200 g Complete Freund’s Adjuvant (CFA), 100 g myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, and 200 ng Bordetella pertussis toxin (lot# 181236A1, List Biological Laboratories …

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

… Clostridium difficile toxin B (TcdB) (List Biologicals ): Spin down toxin tube for 30-45 s at 20,000 g. Reconstitute 20 g of TcdB into 100 L of sterile double-distilled water to obtain a 200 g/mL stock solution. Store at 2-8 C (see Note 5 ). …

• Product #155 – Toxin B from Clostridium difficile