Citations

Induction of EAU and treatment:

Each C57BL/6J mouse was immunized subcutaneously in one rear footpad and the tail head, and rump (total of 200l) with an emulsion containing 300µg of human IRBP₁₂₀… Simultaneously, the mice received 1µg of Bordetella pertussis toxin (List Biological Laboratories, Campbell, CA, USA) intraperitoneally. Then the mice were randomly divided into a chrysin-treated group (n=30 mice) and a control group (n=30 mice). …

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:

…immunized with 200 µg MOG 3355 peptide emulsified in complete Freund’s adjuvant (Sigma) containing Mycobacterium tuberculosis H37 RA (final concentration 2 mg/ml) on day 0, and ip administered with 200 ng pertussis toxin (List Biological Laboratories)…

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)

Mucosal immunization

For intranasal (i.n.) administration, immunization cocktails were made by mixing the indicated proteins with PBS. Each dose (20 μl total, 10 μl per nostril) contained either CTB-MPR (35 μg ≡ 12 μg MPER ≡ 2 nmol MPER) with cholera toxin (CT, 1 μg, List Biological Laboratories), VLP preparation (1.1 mg total protein containing 48 μg dgp41 and 48 μg Gag, equivalent to 2 nmol and 1 nmol, respectively) with either CT (1 μg), …

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

Reagent:  1 nM cholera toxin (List Biological Laboratory, Campbell, CA)

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

EAE induction and LPS instillation:

Active EAE was induced by immunizing the mice with MOG₃₅₅₅ peptide (100 µg) emulsified in complete Freunds adjuvant (Sigma) containing heat-killed Mycobacterium tuberculosis H37Ra (200 µg per mouse, DIFCO Laboratories) on day 0, and intraperitoneal injection of 200ng pertussis toxin (PTx; List Biological Laboratories) on days 0 and 2. To induce T_h17-mediated passive EAE, CD4⁺ T cells, obtained from 2D2 mice, were polarized with rIL-6 (20ng ml¹), rIL-23 (10ng ml¹), rTGF- (3ng ml¹) and anti-IFN- antibody (4 g ml¹) on a plate coated with anti-CD3 and anti-CD28 antibodies for 5 days and adoptively transferred to sublethally irradiated (450 rads) WT and Atg7 CKO mice. PTx (200ng) was intraperitoneally administrated on days 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)

CTb into NAc shell:

We unilaterally injected 40 nl of 1% CTb (List Biological Laboratories) into the NAc shell over 4 min with the needle left in place for an additional 2 min.

CTb injection site verification:

Immunohistochemical procedures were based on our previously published work…We selected a 1-in-4 series of sections from the NAc shell of each rat, and immunofluorescence was used to determine CTb injection sites. We repeatedly rinsed free-floating sections in PBS (3 10 min washes) and incubated them for 2 h in PBS containing 0.5% Triton X-100 (PBS-tx) with 10% normal horse serum (NHS). We then incubated all sections for at least 48 h at 4°C in goat anti-CTb primary antibody (1:5000, List Biological Laboratories, 703),

Fos protein and CTb double labeling:

We processed a 1-in-4 series of vmPFC, PVT, BLA, and vSub for immunohistochemical detection of Fos protein and CTb…We then incubated sections for at least 48 h at 4°C in PBS-tx with 2% NHS, rabbit anti-c-Fos primary antibody (1:8000, Cell Signaling Technology, Phospho-c-Fos, 5348S) and goat anti-CTb primary antibody (1:5000, List Biological Laboratories, 703).

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt
• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

EAE:

… injected subcutaneously into the tail base. Immediately thereafter, they received 200 ng pertussis toxin (List Biological) into the foot pad [21]. The animals were examined daily for signs of EAE. Clinical signs were evaluated as …

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 Laboratories, 104, 1 – 1.2%. Anti-CTB, List Biological Laboratories, 703, …

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt
• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

Bacterial Strains and Reagents:

… Activated protective antigen was procured form List Biological Laboratories (USA). …

2.6. Binding to LF and Native-PAGE

For the confirmation of functional activity of the expressed PA, 2g of trypsin digested PA was incubated with 2g of LF at room temperature for 30 minutes. The samples were run on 4% native polyacrylamide gel at 4C and visualized by staining the gel with coomassie brilliant blue.

2.7. Animal Immunization

Six female Balb/c mice were procured from Institutional Animal Care Center, DRDE, and were provided with food and water ad libitum. Each mouse was immunized subcutaneously with 10g PA adsorbed on aluminium hydroxide adjuvant on days 0, 14, and 28. Mice receiving only PBS were taken as control. All mice were bled prior to first immunization on day 0 and on 35th day. Serum was separated and stored at 20C until further use.

2.8. Enzyme Linked Immunosorbent Assay

To test the reactivity of PA immunized sera, indirect ELISA was performed by coating 1gmL¹ PA in ELISA plate overnight at 4C. After blocking with 5% skim milk for one hour, serial twofold dilution of PA immunized sera starting from 1:1000 was added and incubated for one hour at room temperature. …

2.9. Cell Culture

Mouse macrophage cell line J774.1 was procured from National centre for cell sciences, Pune, India and was maintained in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 100UmL¹ penicillin and streptomycin as antibiotics. One day before the experiment 4 10⁴ cells were seeded in a 96 well plate and incubated at 37C in presence of 5% humidified CO₂. The next day spent media was replaced by a fresh medium containing gradient concentration of soluble PA and native PA of B. anthracis from List Biologicals (0.005gmL¹ to 5gmL¹) and was incubated at 37C along with 0.125gmL¹ LF. After 4 hours, 20L of 5mgmL¹ MTT (3-(4,5 dimethylthiazol-2 yl)-2,5 diphenyl tetrazolium bromide) was added to each well and further incubated for 30 minutes.

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis

… Purified LPS was purchased from List Biologicals (San Jose, CA, USA) and was dissolved in endotoxin-free water. …

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/