Citations

4.3. EAE induction

EAE was induced using 200 μg/dose of MOG(35–55) peptide (MDBiosciences, Redoxis exp.) or 100 μg MOG(35–55) (Genemed Synthesis, Inc., UCSF exp.) emulsified with complete Freunds Adjuvant (CFA) containing IFA (Difco) with M. Tuberculosis H37RA (300 μg or 800 μg H37RA/dose (BD Difco) in the Redoxis experiment (Exp. 1) or UCSF experiment (Exp. 2), respectively). Mice were anaesthetized with isoflurane and the emulsion was injected subcutaneously (volume 100 μl or 200 μl in the Redoxis experiment or UCSF experiment, respectively) in the flank. Pertussis toxin (Ptx, 200 ng/dose, MerckMillipore or List Biological) was administered intraperitoneally (i.p.) 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)

… Mycobacterium tuberculosis (strain H37Ra; Cat# 231141, Becton, Dickinson and Company, Franklin Lakes, USA), followed by intraperitoneal administration of 400ng of Pertussis toxin (List Biological Laboratories, Cat# 181, Campbell, USA) on day 0 and after 48?h (Bonfiglio et al., 2017). …

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

Reagents and Biological resources

… E. coli O111:B4 Lipopolysaccharide (LPS) was purchased from List Biological Laboratories (catalog #201). …

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

Retrograde Tracing

Injections of CTB (List Biological Labs, Campbell, CA) into the NAcc (A: 1.30, L: ±0.85, D: -3.80), CPVM (A: 0.26, L: ±1.25, D: -3.0), LHb (A: -1.4, L: ±0.75, D: -3.90), VTA (A: -3.3, L: ±0.4, D: -4.0), and DRN (A: -2.24, L: -1.1, D: -2.85 with 20° angle) were performed stereotaxically in WT and 15q dup mice: 50 μL of 0.5% CTB solution dissolved in saline was injected using a glass micropipette. The mice were anesthetized 5 days post-CTB injection, with an intraperitoneal injection of an anesthetic combination comprising 0.3 mg/kg medetomidine, 4.0 mg/kg midazolam, and 5.0 mg/kg butorphanol. Then, they underwent cardiac perfusion fixation with 4% PFA in 0.01 M PBS. The post-fixed brains were rinsed with saline and cryoprotected overnight with 30% sucrose in PBS at 4°C. Brain sections (50 μm) were cut using a sliding microtome (HM430; Thermo Fisher Scientific, Waltham, MA, USA). Immunostaining was performed as previously described.

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

2.2. Experimental autoimmune encephalomyelitis (EAE) induction

6–7 weeks old C57BL/6 female mice were immunized with an emulsion containing 200 μg of purified myelin oligodendrocyte glycoprotein peptide (MOG35–55) in normal saline (0.9% NaCl) and an equal volume of complete Freund adjuvant (Betcon, Dctinson and company, sparks MD, 21152 USA). 0.1 ml volume of the inoculum was injected subcutaneously (S·C) on day 0 into each hind limb. 200 ng pertussis toxin (List biological laboratories INC) was diluted in 0.1 ml normal saline (0.9% NaCl) and was injected I·P immediately after S·C injection of MOG35–55 emulsion 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)

Serological analysis of B. pertussis-specific antibodies

On the day of mouse processing (euthanasia and dissection), a cardiac puncture was performed on each mouse. Approximately 1 mL of blood was removed from each mouse. The samples were placed into serum separator tubes (Cat. Number: 365967; BD) centrifuged for 2 min at 14,000 × g. Serum was collected and stored at −80°C until analyses were performed. Serological responses specific to B. pertussis antigens were quantified by enzyme-linked immunosorbent assay (ELISA). High-binding microtiter plates were coated with pertussis toxin (PT) (50 ng/well) (PT#180, LIST Biologicals), filamentous hemagglutinin (FHA) (50 ng/well) (Enzo Life Sciences), or PRN (50 ng/well) (PRN#187, LIST Biologicals) as described in Boehm et al. (34). …

• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #187 – Pertactin from B. pertussis (69 kDa Protein)

… Unless otherwise specified, mice were sensitized by a single intraperitoneal injection of 250 µg of OVA (Sigma-Aldrich, A5478) and 5 µg of CT (List Labs; 100B) in phosphate-buffered saline (PBS; Sigma-Aldrich, P4417-100TAB). Epicutaneous sensitization was performed by applying 200 µg of OVA after shaving or tape stripping the lower back …

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

Cell Culture

MCF-10A cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)- F12 (Welgene, LM002-04) supplemented with 5% horse serum (Invitrogen, 26050-088), epidermal growth factor (Peprotech, AF-100-15) at 20 ng/ml, insulin (Gibco, A11382II) at 10 μg/ml, hydrocortisone (Sigma, H0888) at 0.5 μg/ml, and cholera toxin (List Biological Laboratories, 100B) at 100 ng/ml. HEK293T, MDCK, HFE-145 (gastric epithelial) cells and PANC-1 (pancreatic adenocarcinoma) cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) (Gibco, 12483020). AGS, MKN-45 (both from gastric adenocarcinoma), and BCPAP (thyroid carcinoma) were cultured in RPMI medium supplemented with 10% FBS.

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

EAE

… The mice were s.c. immunized with 100 µg of MOG (Sigma-Aldrich) emulsified in CFA (BD, Sparks, MD) plus 500 µg of Mycobacterium tuberculosis H37Ra (BD). At the times of immunization and 2 d later, the mice were i.v. injected with 400 ng of pertussis toxin (List Biological Laboratories). Disease severity of EAE was monitored using the criteria as previously described (26). …

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)

Induction and scoring of Experimental Autoimmune Encephalomyelitis (EAE) mice

Female mice were subcutaneously injected with an emulsion of 50 μg myelin oligodendrocyte glycoprotein (MOG) amino acids 35-55 (Sheldon Biotech, Montreal, QC) peptide in incomplete Freund’s adjuvant (Sigma) supplemented with 1 mg of mycobacterium tuberculosis (CFA) (Difco). 400 ng of pertussis toxin (List Biologicals) was administered intraperitoneally on days 0 and 2 post-immunization. For ligand treatment, 40 μg of purified protein was administered intravenously once every three days for the duration of the experiments. …

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)