Citations

2.2.1. Induction of EAN

The P0180–199 peptide was synthesized by Ontores Biotech (SSKRGRQTPVLYAMLDHSRS, >98% purity; Hangzhou, China). Freund’s incomplete adjuvant and Heat killed Mycobacterium tuberculosis (strain H37RA) were purchased from BD Difco (#263910, #231141; MI, USA). Pertussis toxin (PTX) was purchased from List Biological Laboratories (#181; CA, USA). Briefly, mice were anesthetized with isoflurane before immunization. PTX solution (1.6 μg/mL, 1.2 μg/mL and 1.2 μg/mL in 250 μL volume each were intraperitoneally administered on Day −1, 1, and 3, respectively. The P0180–199 peptide of 2 mg/mL was emulsified in Freud’s complete adjuvant (20 mg/mL solution of Mycobacterium tuberculosis in Freund’s incomplete adjuvant). The emulsive inoculum of 50 μl was injected subcutaneously into mice on days 0 and 8 [18].

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

… Mice then received an intraperitoneal injection of 180 ng freshly diluted pertussis toxin (List Biological Laboratories or MilliporeSigma) 2 to 5 hours later and again in 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)

Botox injection

Botox (Botulinum Neurotoxin Type A, List Labs #130A) was reconstituted to a stock solution (20 ng/µl) with distilled water containing 1 mg/ml bovine serum albumin. To make injection solution, the stock was diluted with phosphate buffered saline to 10 pg/µl. Mystacial pads were subcutaneously injected with 1 µl of solution via microliter syringe (Hamilton). At first injection, mice stopped whisking within hours, and paralysis from a single injection lasted 7 days with full whisking recovering within 2 weeks. To achieve continuous paralysis across the Botox imaging period, we injected a supplemental 50% dose once per week. We inspected mice daily to confirm the absence of whisking.

Experimental Autoimmune Encephalomyelitis

Active EAE experiments were conducted via immunization of 8-12 week old female C57BL/6 mice with MOG35-55 (Alpha Diagnostic), as previously published12,16,19,20 30 but with one 400ng intraperitoneal injection of pertussis toxin (List Labs) on day 2. Passive EAE experiments were performed as per previously described protocols 12,15,19 32 with the following modifications. Active EAE was first induced via immunization with MOG35-55 and one 400ng intraperitoneal injection of pertussis toxin (List Labs) on day two. Splenocytes 2 were isolated from immunized female mice on day 8, cultured in presence of TH1/TH17 3 polarizing conditions, and adoptively transferred into naïve MCAM KO or WT recipient 4 female mice on day 12. The recipient animals received a 200ng intraperitoneal injection of 5 pertussis toxin (List Labs) on day 14.

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)

BoNT/A

BoNT/A(List Biological Labs, 130B) was injected (25 pg μl−1, i.d., five neighbouring sites injected with 20 µl) into the skin three days and one day before tumour inoculation (defined as prophylactic). BoNT/A (25 pg μl−1; i.d., five neighbouring sites injected with 20 µl) was injected around the tumour one day and three days after the tumour reached a volume of around 200 mm3 (defined as therapeutic) in other groups of C57BL/6J mice.

• Product #130 – Botulinum Neurotoxin Type A from Clostridium botulinum

… Mycobacterium tuberculosis, and was injected i.p. on days 0 and 2 with 400ng pertussis toxin (List Biological Laboratories, Inc. Campbell, CA), as described [16]. …

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)

Reagents

… Bacillus anthracis PA was obtained from List Biological Laboratories. …

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

… A 10× solution of the lethal factor (LF, List Biological Labs) in medium was prepared at 5 µg/mL, and 10 µL of the 10× stock was added to the well to achieve a 0.5 µg/mL …

Author did not specify which Lethal Factor (LF) was utilized; List Labs provides the following LF:

• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

2.4. Bone Marrow Derived Mast Cells

Bone marrow-derived mast cells (BM-MCs) were in vitro differentiated into a mucosal-like phenotype as described previously [13]. Briefly, bone marrow cells were collected from femurs and cultured in DMEM with glucose and L-glutamine, supplemented with 10% fetal bovine serum, penicillin/streptomycin, and sodium pyruvate (Biowest, Nuaillé, France), plus stem cell factor, IL-3, IL-9 and TGF-β (Peprotech, Rocky Hill, NJ, USA), for a minimum of 4 weeks and a maximum of 8 weeks before being used. For activation through cross-linking of the IgE receptor, MCs (1 × 106 cells) were sensitized with serum from BALB/c mice orally sensitized with 5 mg of SM, CN, and WP plus 10 μg of cholera toxin (List Biologicals, Campbell, CA, USA) [14]. After washing, MCs were activated with samples in HEPES degranulation buffer. To study the activation through an IgE independent pathway, non-sensitized MCs were also stimulated with samples. β-hexosaminidase was detected by an enzymatic colorimetric assay [15]. The percentage of β-hexosaminidase release was calculated considering the total β-hexosaminidase content.

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

Multiplex bead assay.

The specificities and avidities of clonal IgG antibodies in culture supernatants were determined in a multiplex bead assay (Luminex Corp.) as described previously (18, 36), with modifications. … Antigens and controls included BSA, … tetanus toxoid from Clostridium tetani (List Biological Laboratories), …

• Product #191 – Tetanus Toxoid from Clostridium tetani