Citations

EAE Induction

EAE induction was based on a published protocol [24]. Murine myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide (MOG35–55, MEVGWYRSPFSRVVHLYRNGK) was synthesized with >99% purity (SciLight Biotechnology, China). C57BL/6 mice were injected with 200 μg MOG35–55 emulsified in 100 μg of complete Freund’s adjuvant (CFA, Sigma) and an additional 400 μg Mycobacterium tuberculosis H37RA (BD Biosciences) by subcutaneous injection into the flanks. These mice were also given 400 ng pertussis toxin (List Biological Laboratories) in 100 μl phosphate buffered saline (PBS) on the same day and 2 days later by intraperitoneal injection.

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)

… In Experiments 4 and 5, for visualization of CTb, all section sets were preincubated in 0.02 M PBS containing 3% normal goat serum, 0.2% Triton X-100 and a polyclonal rabbit anti-CTb primary antibody (List Biological Laboratories, USA) diluted to 1:40,000 …

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

… Pertussis toxin was purchased from List Biological Laboratories, Inc. (Campbell, CA, USA) …

EAE induction and pharmaceutical treatment

To induce EAE disease, C57BL/6 male mice (6–8 weeks) received s.c. injections of complete Freund’s adjuvant (CFA) containing Mtb (200 μg) and MOG35–55 (100 μg) at 0 days post immunization (dpi). Pertussis toxin (200 ng per mouse) was administered through intraperitoneal (i.p.) injection at 0 and 2 dpi. GA or sGA (1 mg kg−1, 10 mg kg−1, or 100 mg kg−1) were administered through i.p. injection at 2 dpi (one-time-dosing) or 2 and 5 dpi (two-time-dosing) (n = 4 to 8). …

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)

Chemicals and reagents

… Toxins (product numbers) purchased from List Biological Laboratories were: EF (178A), diphtheria toxin (150), Clostridium septicum alpha toxin (116L), C. perfringens epsilon toxin (126A), C. difficile toxins A (152C), B (155B), and binary toxin subunit A(157A), Pasteurella toxin (156), pertussis toxin (181), botulinum neurotoxins A (612A) and B (622A) (heavy chains), and cholera toxin (100B). …

• Product #178A – Anthrax Edema Factor (EF), Recombinant from B. anthracis
• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae
• Product #152C – Toxin A from Clostridium difficile
• Product #155 – Toxin B from Clostridium difficile
• Product #156 – Pasteurella multocida Toxin
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #612A – Botulinum Neurotoxin Type A Heavy Chain, Recombinant, Binding Domain
• Product #622A – Botulinum Neurotoxin Type B Heavy Chain, Recombinant, Binding Domain
• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

… On the day of and 2 d after immunization, 200 ng of pertussis toxin (List Biological) was injected intravenously …

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)

Experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis was induced using methods described previously (Lu et al., 2020). Specifically, complete Freund’s Adjuvant (CFA) was generated by adding heat killed Mycobacterium tuberculosis H37 RA (MT; Difco, No. 231141) with incomplete Freund’s adjuvant (Difco, No. 263910) to achieve a final concentration of 5 ​mg/ml. Equal volumes of MOG35-55 (Anaspec Inc.; 4 ​mg/ml) and the adjuvant mix were vortexed for 45 ​min. Mice were given four subcutaneous injections of MOG35-55/adjuvant emulsion into the hind flanks. Finally, mice received an i.p. injection of sterile PBS containing 400 ​ng of pertussis toxin (List Biological Laboratories) on days 0 and again on day 2 post immunization. Disease was scored by raters blinded to genotype as described previously (Lu et al., 2020). Control (CFA) C57BL/6 mice were immunized with CFA in the absence of antigen.

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) solution (Cat#104, List Biological, Campbell, CA) bilaterally into the DIAm (Prakash et al., 1993; Prakash et al., 2000; Rana et al., 2019b) An anti-CTB (Goat polyclonal, Cat # 703, Lot # 7032A9, 1:5000; List Biologicals) was used to label PhMNs …

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

Antigens for immunization and laboratory assays.

Purified Vi polysaccharide and DT-conjugated Vi polysaccharide (Vi-DT) (both gifts from the International Vaccine Institute, IVI, in South Korea), and CT (CT; List Biological Laboratories Inc., Campbell, CA) were used for immunizations. Each mouse was transcutaneously immunized with 25 µg of Vi, 25 µg of Vi in Vi-DT conjugate, or 10 µg of CT alone or as an adjuvant with Vi or Vi-DT. Typhim Vi (Sanofi Aventis, Bridgewater, NJ), CT, and keyhole limpet hemocyanin (KLH; Sigma Chemical Co., St. Louis, MO) were used for detection of immunologic responses.

Immunization regimen and schedule.
Five different groups of mice (N = 10–15 each) were immunized transcutaneously as previously described13 with CT, Vi, Vi conjugated with DT (Vi-DT), or Vi or Vi-DT with CT as an adjuvant.

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

… Injection of retrograde tracers into the SNc and VTA. Two retrograde neural tracers, Fluoro-gold (FG; Fluorochrome, Denver, CO, USA) and cholera toxin subunit B (CTb; List Biological Laboratories, Campbell, CA, USA) were prepared …

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

Materials

… Cholera toxin (CT) (Lot#10067A1) was obtained from List Biological Laboratories, Inc. (Campbell, CA) …

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