Citations

Chemicals, Peptides, and Recombinant Proteins

Pertussis toxin List Biological Laboratories Cat# 180

EAE

Mice were immunized subcutaneously in the flanks with an emulsion containing MOG35–55 (80 μg/mouse) and M. tuberculosis H37Ra extract (3 mg/ml, Difco Laboratories) in CFA (100 μl/mouse). Pertussis toxin (100 ng/mouse, List Biological Laboratories) was administered intraperitoneally on days 0 and 2. Mice were monitored and assigned grades for clinical signs of EAE as previously described (Xiao et al., 2015). To evaluate the suppressive ability of Tim-1+ B cells, purified total B cells or Tim-1+ B cells were transferred i.v. into WT recipients. Hosts were then immunized to induce EAE.

• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer

… To deplete CCR2+ monocytes, 5g per kg DT (List Biological Laboratories) was injected into all mice at day 10, 13, 16 and 19 …

• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae

EAE induction and scoring

Mice were immunized subcutaneously at the tail base with 50 μg MOG35-55 peptide (GenScript) emulsified in complete Freund’s adjuvant (CFA, BD Biosciences) supplemented with 10 mg/ml Mycobacterium tuberculosis H37RA (BD Biosciences). In addition, mice were intraperitoneally injected with 100 ng of pertussis toxin (PTx) (List Biological Labs) on day 0 and day 2 post-immunization. …

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)

Toxin Neutralization Assay (TNA)
The functional ability of antibodies from vaccinated cattle and controls to neutralize anthrax lethal toxin was assessed using an in vitro TNA, as previously described by Ndumnego et al. [16]. The TNA was determined with J774A.1 mouse macrophage cells (ECACC cat no 91051511) exposed to anthrax lethal toxin in the presence of antibodies. Briefly, 1.0 × 105 J774A.1 macrophages per well in Dulbecco’s Modified Eagle Media (DMEM) and 10% foetal bovine serum (FBS) were incubated at 37 °C and 5% CO2 overnight in 96-well cell culture plates. Sera from each animal were 2-fold serially diluted in duplicates with the starting dilution of 1:50 in DMEM formulated with 5% FBS containing PA (500 ng/mL) and LF (400 ng/mL) (List Biological Laboratories Inc, Campbell, CA, USA) for 1 h at 37 °C and 5% CO2 before addition to the overnight-cultured confluent J774A. …

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

Detection of anthrax-specific antibody levels

Anthrax-specific antibody levels against protective antigen were measured by ELISA12. High binding ELISA plates were coated with 100 ng per well of anthrax protective antigen (List Biological Laboratories Inc., Campbell, CA, USA) in carbonate buffer, pH 9.6 and incubated at 37 °C for 1 h, then overnight at 4 °C. …

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

… Seventy two hours following adenoviral treatment, cells were pretreated with 50uM Z?VAD?FMK (BD Biosciences) for 1 h prior to stimulation with 100 ng/ml recombinant human TNFa (Peprotech), 10ng/mL LPS (List Biological Laboratories, Campbell, CA, 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/

… The following oligosaccharides were used: … C. difficile toxin A was purchased from List Biological Laboratories. …

Transepithelial Electrical Resistance Measurements and Cell Integrity
T84 cells on Transwell inserts were cultured overnight in DMEM/F-12 Ham medium containing 2mM L-glutamine. The apical media was replaced with test sample and incubated for 2 h at 37 °C after which C. difficile toxin A was added to 10–100 ng/mL. During each experiment duplicate inserts were used for each condition. Variations in toxin specific activity precluded the use of a constant amount. Therefore, concentrations of the toxin were added to T84 cells such that after approximately 20 h of incubation under control conditions only 20% of baseline TEER remained. Experimental T84 responses were normalized to responses in the presence of media alone (0% protection) and MRS at 20% (v/v) (100% protection) as a reference positive control. MRS was previously shown to have protective properties against cell damage caused by toxin A (30). …

• Product #152C – Toxin A from Clostridium difficile

SM22α-hDTR mice

All animal studies were undertaken under UK Home Office licensing and approved by the Animal Welfare Ethical Review Body of the University of Cambridge. C57BL6/J SM22α-hDTR mice were generated and genotyped as previously described [13]. Male and female SM22α-hDTR mice and wild type littermates were treated as indicated with intraperitoneal doses of 5 ng/g body weight diphtheria toxin (LIST biological laboratories, 150) diluted in 0.9% saline and 0.2% BSA.

• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae

Antibodies used in this study

… CTb, Goat, 703, 1:1000, List Biological.

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

Generation of EAE

Before the induction of EAE, all mice were kept in the same environment for at least 10 days to reduce microbiome differences. For MOG35-55 peptide immunization, mice were lightly anesthetized with isoflurane and subcutaneously (s.c.) immunized with 0.2 mg MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK, Bio-synthesis) in 0.1 ml PBS plus 0.1 ml complete Freund’s adjuvant (CFA) (Thermo-Fisher) supplemented with 0.4 mg M. tuberculosis H37 Ra (BD). On day 0 and 2 post immunization, mice were intravenously (i.v.) injected with 0.2 μg Pertussis toxin (List Biological Laboratories) in 0.2 ml PBS. …

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)