Bacterial Toxin Research Citations

Cell culture:

Bone marrow-derived macrophages (BMDM) were prepared by harvesting marrow from femurs and tibias of 129+/+ and Nlrp1b2/2 mice. Marrow was cultured for 7 d in IMDM containing 15% L cell-conditioned medium, 10% FBS, 2 mM L-glutamine, 150 U/ml penicillin, 150 mg/ml streptomycin, and 57.2 mM 2-ME (all from Life Technologies). BMDM were incubated in OPTIMEM plus Glutamax (Life Technologies) containing varying concentrations of LT (List Biological Laboratories, Campbell, CA) for 4 h, and cell viability was assessed with the WST-1 reagent (F. HoffmannLa Roche, Basel, Switzerland). Peritoneal macrophages were elicited from wild-type, Nlrp1b2/2, Nlrp32/2, and Casp-12/2 mice with an i.p. injection of 4 ml 4% thioglycollate in PBS. Seventy-two hours after injection, the peritoneal cavity was lavaged with DMEM, and cells were plated and incubated for 1 h. Adherent cells were then washed twice with cold PBS and cultured in DMEM containing 10% FBS, 2 mM L-glutamine, 150 U/ml penicillin, 150 mg/ml streptomycin, and 57.2 mM 2-ME (all from Life Technologies). For LT treatments, macrophages were primed overnight with 100 ng/ml ultrapure LPS (Invitrogen Life Technologies), washed with PBS, and incubated with OPTIMEM plus Glutamax containing either PBS or 1 mg/ml LT for 6 h. …

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

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

Materials:

… Ultrapure LPS (TLR4 ligand) isolated from Salmonella minnesota R595 was obtained from List Biological Labs (Campbell, CA). …

Cell culture and ELISA:

… Monocytes were isolated with .95% purity, based on CD14 positivity in ow cytometric analysis and were maintained in RPMI 1640 supplemented with 10% FCS. Cells were incubated with TLR ligands PAM2, PAM3, and LPS, or with M. tuberculosis whole cell lysates for 24 h. …

• Product #434 – ULTRA PURE LPS from Salmonella minnesota R595 (Re)

EAE immunization:

Mice were immunized s.c. over four sites on the ank with 100 ml of an emulsion containing 200 mg myelin oligodendrocyte glycoprotein (MOG) 3555 (CS Bio) in PBS and an equal volume of CFA (containing 200 mg heat-killed Mycobacterium tuberculosis, Jamaica strain). Mice also received 200 ng pertussis toxin (List Biological Laboratories) injected i.p. at the time of immunization and 48 h later. …

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)

EAE induction:

For active EAE, B6 mice were s.c. immunized with 100 mg MOG3555 peptide (MEVGWYRSPFSRVVHLYRNGK) (Biopolymers Laboratory, Harvard Medical School) emulsied in CFA solution (4 mg/ml mycobacteria in IFA). On the day of immunization and 2 d after, mice received 200 ng pertussis toxin (List Biological Laboratories) in 100 ml PBS i.v. …

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)

… A glass micropipette (20-30 m id) filled with 1% low salt CTB (List Biological Laboratories, Campbell, CA) in distilled water was stereotaxically lowered into part of the left retrosplenial cortex or presubiculum, where BDA-labeled terminals were densely distributed in the …

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

EAE:

… Intraperitoneal injections of 500 ng pertussis toxin (List Biological Laboratories, Campbell, CA) dissolved in 200 µL PBS were administered immediately after the first MOG injection on Day 0 and 48 hours later. …

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)

Materials:

… Shiga toxin Stx2 was purchased from List Biological Laboratories, Inc. (Campbell, CA). …

Milk Inhibits Ricin but Not Shiga Stx2 Toxin:

To demonstrate that inhibition of toxin activity is specific to ricin, we also evaluated the effect of milk on the Stx2 produced by enterohemorrhagic strain Escherichia coli O157:H7. This bacterial toxin acts similarly to ricin in inactivating ribosome proteins, and similar to ricin, is composed of two subunits. However, in contrast to ricin, the Stx2 B chain binds specifically to the cell surface receptor glycolipid globotriasylceramides Gb3 and Gb4. In addition, the two toxins seem to use different endocytic pathways (21).
To test the specificity of milk inhibition and antibody neutralization, we compared the inhibition of GFP expression in Vero cells by Stx2 (1 ng/ml) and ricin (1 ng/ml) in the presence and absence of milk or antibodies. Fig. 6 shows that milk had the same protective effect in inhibiting ricin as did the anti-ricin antibody, as indicated by GFP fluorescence intensity levels. The results were nearly the same for the milk samples with and without ricin, indicating that 1% milk was fully effective at inhibiting ricin toxicity. 

List Labs has discontinued Product #162 (Lyophilized) and has replaced Product #162 with Product #162L (Liquid):

• Product #162L (Liquid) – Shiga Toxin 2 from Escherichia coli

Sensory tracing:

Three days prior to sacrifice, all 17 remaining animals in Experiment 1 and the remaining 19 animals in Experiment 2 that did not receive CST tracing, were reanesthetized with isoflurane and 2% O₂, and the sciatic nerve was isolated, crushed, and injected with 5L of cholera-toxin beta (CT; ListBiological Laboratories, Inc., Campbell, CA). The needle was left in place briefly before the skin was closed with wound clips. The animals were given buprenorphine and gentamicin as above.

Tissue collection and histology:

… CT-labeled sensory axons originating from L3L5 were identified by immunohistochemistry for CT followed by ABC staining. An entire set of slides containing all cases from Experiments 1 and 2 that received injections of CT were processed at the same time. Tissue sections were washed with Tris-buffered saline with 2% Triton (TBS-T), treated with 2:1 chloroform:methanol to remove lipids for 1h, rewashed, and treated with 10% methanol, then rinsed with 30% H₂O₂ in distilled H₂O for 1h to quench endogenous peroxidase activity. The tissue was then washed with TBS-T and blocked overnight with 5% normal rabbit serum. This was followed by incubation with goat anti-CT (1:20:000; List Biological Laboratories, Campbell, CA) for 60h, …

Author did not specify which CTB was utilized.  List Labs provides Product #103B (Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae)  and Product #104 (Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt).

• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

… LPS(lipopolysaccharide) List Biological …

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/

Surgical Procedure:

… Injections were made with an adapted Hamilton syringe of 10 µl, the plunger of which was moved by a programmable microsyringe pump (flow 200 nl/min), connected by way of thin tubing (inner diameter, 0.1 mm) in which the movement of an air bubble was monitored to enable control of the injected volume. The injectate contained a mixture of 1 part 1% CTb (low salt; List Biological Laboratories, 1% w/v in 0.2 M phosphate buffer, pH 7.4) and 4 parts RV (Ruigrok et al., 2008; Prevosto et al., 2009). …

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