Bacterial Toxin Research Citations

Active EAE induction:

… A booster immunization was applied subcutaneously on day 7 over contralateral lymph nodes. Pertussis toxin (500 ng/mouse) (List Biological Laboratories) was injected intraperitoneally on days 0 and 2 (54). …

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)

… Wild-type and Plgf-KO mice on the C57BL/6N background were subcutaneously immunized with 200 µg of MOG3555 peptide (MEVGWYRSPFSRVVHLYRNGK) emulsified in 100l CFA (Sigma-Aldrich) in the abdominal flank. On days 0 and 2, the immunized mice received additional intraperitoneal injections of 200ng of pertussis toxin (List Biological Labs). …

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)

… antiCTB at 1:10,000 (List Biological Laboratories, Campbell, CA). …

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

… An anti-CTB (Goat polyclonal, Cat #703, Lot # 7032A9, 1:5000; List Biologicals) was used to label PhMNs. …

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

Reagents and antibodies

… For cell culture experiments, Val-boroPro was resuspended in DMSO containing 0.1% TFA to prevent compound cyclization. Anthrax Lethal Factor (Catalog #172B) and PA (Catalog #171E) were purchased from List Labs, …

LDH cytotoxicity assays

For LDH release experiments involving primary macrophages, mBMDM and rBMDM were isolated from the femurs and tibias of 79-week-old male and female mice and 712-week-old male rats. 110⁷ cells were plated on 10cm Petri dishes. mBMDMs were differentiated in DMEM with 10% FBS and 20% L Cell medium for 6 days and rBMDMs in 30% L Cell medium for 79 days. Macrophages were scraped and reseeded at 0.2510⁶ cells/well in 12-well plates in Opti-MEM and incubated overnight before treatment. Cells were then treated as indicated with compounds or LT. …

In vitro and in cell LF cleavage assays

HEK 293T cells were seeded at 0.510⁶ cells/well in six-well plates and transiently transfected with 2g of the indicated plasmids using FuGENE HD. For the in vitro assay, lysates were harvested after 48h and normalized to 1mg/mL using the DC Protein Assay kit (Bio-Rad). These lysates were then incubated with LF (1g/mL) for the indicated time periods at 25C quenched by boiling with 2SDSPAGE sample buffer at the indicated times, separated by SDSPAGE, immunoblotted, and visualized using the Odyssey Imaging System (Li-Cor). For the in cell assay, after 48h after transfection cells were treated with LT (1g/mL, 6h) before lysates were harvested for immunoblotting as described above.

• Product #172B – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Antibodies and reagents:

…Pertussis toxin (PTx) and enzymatically inactive mutant PTx (PTx_mut) were obtained from List Biological Laboratories. 

• Product #184 – Pertussis Toxin Mutant

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)

… CTb List Biological goat polyclonal, Cat.# 703, lot# 115037, …

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

For treatment with purified CTX, mice were treated by oral gavage with 10 µg purified CTX (List Biological Laboratories) in 0.05 ml LB broth or by mixing 10 µg purified CTX with 0.05 ml LB broth containing the indicated V. cholerae inoculum.

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

LPS:

Ultrapure-grade LPS prepared from Escherichia coli 0111:B4 (List Biologicals, Campbell, CA) was reconstituted in sterile saline at 10g/ml and stored in aliquots at 20C until use in experiments. The specific bioactivity for the individual lot (4216A2) used for this work was not available, but the predicted activity provided by the vendor based on a similar lot was 6.7510⁶ EU/mg (communication from List Biologicals).

In vitro LPS challenge:

Whole blood was collected from animals into sodium heparin tubes (Beckton Dickinson, Caanan, CT) to prevent coagulation. Aliquots (200l) of each blood sample were dispensed into 96-well polypropylene plates and placed at 37C and 5% CO₂ for 30min. LPS diluted in sterile saline or saline only (control) was added to sample wells (10l volume) to achieve final LPS concentrations ranging from 0 to 10g/ml. The plates were incubated for an additional 5h at 37C and then centrifuged at 4C for 10min at 600g to pellet the cells. Plasma collected from each well was stored at 80C until analyzed. The concentration of TNF in each plasma sample was measured by ELISA using the Monkey TNF ELISA kit (U-CyTech, Utrecht, the Netherlands) according to manufacturer instructions. All samples were measured in duplicate.

In vivo LPS challenge and antibody-based modulation:

Animals (n=3/group) were injected IV with 1.0g LPS/kg in a volume of 0.1ml/kg. The LPS was first administered on three different days each separated by a 3-week rest period to establish baseline LPS responses. Prior to the third LPS challenge, blood samples were collected to perform the in vitro LPS challenge (described above) in order to compare in vitro cytokine responses to those induced in vivo. Following another 3-week rest period, animals were injected IV with 0.1, 1, or 10mg/kg of anti-IL-10R1 mAb or with 10mg/kg of the isotype control mAb 2h before a fourth LPS challenge was administered IV. An additional set of control animals was given 10mg/kg anti-IL-10R1 mAb IV and left unchallenged (no LPS). After another 3-week rest period, a fifth and final LPS challenge was performed on all groups.

Blood samples were collected into serum separator tubes (Greiner Bio-One, Monroe, NC) just prior to and 1, 2, 4, 6, and 24h after each LPS challenge to measure concentrations of cytokines (all timepoints) and C-reactive protein (CRP) (pre-challenge and 24h post-challenge only). Blood samples were also collected just prior to and 0.25, 2, 7, 24, 30, 48, 72, 144, 240, 384, and 552h immediately after antibody administration to measure serum anti-IL10R mAb levels. Sera prepared from the blood samples by centrifugation were stored at 80C (for cytokine and CRP assays) or at 20C (for antibody assay) until thawed and analyzed.

• Product #421 – ULTRA PURE LPS from Escherichia coli O111:B4

… LPS was obtained from List Biological Labs (Campbell, USA). …

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/