Citations

2.2. EAE Induction and Scoring

Female mice, aged 6–8 weeks, were immunized subcutaneously with 50 mg MOG35–55 peptide (synthesized by Toray Research Center, Japan) containing 1 mg heat-killed Mycobacterium tuberculosis H37RA emulsified in complete Freund’s adjuvant (Difco, Franklin Lakes, NJ, USA). The mice were injected intraperitoneally (i.p.) with 100 ng of Pertussis toxin (List Biological Laboratories, Campbell, CA, USA) at the time of immunization and 2 days 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)

… Three wild-type mice received stereotaxic injections of FG (2.0%, Fluoro-Gold; Fluorochrome) in the LC and CTB (0.5% Cholera Toxin B subunit, LIST Biological Laboratories) in the PVT. Brain tissues were collected 7 d after surgery and processed for histology. Antibodies against CTB (1:500 dilution; 703/AB_2314252, LIST Biological Laboratories) and FG (1:50 dilution; Fluorochrome) were applied along with anti-TH to identify …

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

EAE induction

Mice were induced with active EAE six to ten weeks post Tamoxifen treatment (after replenishment of IGF-1R+ blood monocytes by natural turnover), as previously described [44]. Briefly, at day 0, mice were immunized with an emulsion of 200 μg MOG peptide (MOG35-55, Genscript, USA) and complete Freund´s adjuvant (Santa Cruz Biotechnology) containing Mycobacterium tuberculosis (Difco) (100 μl/mouse subcutaneously, in the left and right flank and at the tail base). Furthermore, 400 ng pertussis toxin (List Biological Laboratories, Campbell, CA, USA) diluted in sterile DPBS was administered intraperitoneally at day 0 and day 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)

PC-LCA-HPLC

BoNT/A light chain activity was measured after toxin was recovered from reconstituted product using an established PC-LCA-HPLC method. … After washing, digestion buffer (200 μL) containing 0.5 mM zinc acetate, 0.5% Triton X-100 (Dow Chemical Company, Midland, MI, USA), 0.5 mg/mL human serum albumin (HSA), and 2 mM dithiothreitol (DTT) in 50 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (pH 7.4) was added to each well. All concentrations of Triton X-100, HSA, and DTT were optimized to increase assay sensitivity. Plates were then incubated for 30 minutes at 37°C to facilitate toxin reduction. Aliquots (50 μL) of 200 μM SNAPtide 520 substrate (List Biological Laboratories Inc., Campbell, CA, USA) were added to each well, then incubated at room temperature for 72 hours to allow for substrate cleavage. …

• Product #520 – SNAPtide® Peptide Substrate (o-Abz/Dnp) for C. botulinum Type A Neurotoxin

Bphs phenotyping

Bphs phenotyping was carried out as previously described15. Briefly, mice were injected with purified PTX (List Biological Laboratories, Inc.) in 0.025 M Tris buffer containing 0.5 M NaCl and 0.017% Triton X-100, pH 7.6. Control animals received carrier alone. Three days later, mice were challenged by injection with histamine (milligrams per kilogram of body weight [dry weight], free base) suspended in phosphate-buffered saline (PBS). Deaths were recorded at 30 min post-challenge. The results are expressed as the number of animals dead over the number of animals studied.

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)

… After the last day of fentanyl self-administration training, we injected CTb (List Biological Laboratories) into Pir. We injected 40 nl of 1% CTb unilaterally into Pir with the needle left in place for an additional 5 min (Mahler and Aston-Jones, 2012; Marchant et al., 2014, 2016; Venniro et al., 2017b). We counterbalanced injections into either the left or right hemisphere and used a 1.0ml, 32 gauge Neuros Syringe (Hamilton) attached to UltraMicroPump III with SYS-Micro4 controller (World Precision Instruments). The coordinates for Pir were AP, 13.4 mm; ML, 64.3 mm (10° angle lateral to midline); DV, 6.7 mm. These coordinates are based on pilot studies to optimize spread of CTb within Pir and minimize spread to adjacent areas. …

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

Mouse techniques

… For diphtheria toxin receptor (DTR) ablation studies, WT and indicated DTR mice received injection twice intratracheally (i.t.) with 100 ng diphtheria toxin (DT) in 20 μl of sterile PBS or i.p. with 200 ng DT in 100 μl of sterile PBS (150, List Biological Lab) on 1 d before and after metastatic inoculation. Cell of interest depletion was confirmed by flow cytometry. …

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

… Pertussis toxin (180) was from List Biological Laboratories. …

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

Mice and EAE induction

We purchased C57BL/6, (C57BL/6xSJL) F1, 2D2-TCR-Tg, and DO11.10-TCR-Tg mice from the Jackson Laboratory. Mice were maintained in a specific pathogen–free facility and used under protocols approved by the National Institute of Allergy and Infectious Diseases (NIAID) Animal Care and Use Committee (protocol LISB-7E). EAE was induced by immunization of female C57BL/6 mice (10 to 12 weeks old) with 200 μl of emulsion containing 100 μg of MOGp35–55 and 400 μg of Mycobacterium tuberculosis extract H37 Ra (Difco) in Freund’s complete adjuvant (FCA) as previously described (73). At 4 and 24 hours after immunization, mice received 200 ng of pertussis toxin (List Biological Laboratories) intraperitoneally. To induce RR-EAE in (C57BL/6xSJL) F1 mice, we immunized the female F1 mice with 200 μl of emulsion containing 200 μg of MOGp35–55 and 400 μg of M. tuberculosis extract H37 Ra (Difco) in FCA. At 4 and 24 hours after immunization, mice received 75 ng of pertussis toxin intraperitoneally. EAE was scored by the methods previously described (74, 75). …

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)

5.6. The establishment of EAE model in mice

The EAE model was induced and evaluated as previously described48. Briefly, 2 mg/mL of MOG35-55 (Glbiochem, Shanghai, China) was emulsified in complete Freund’s adjuvant (CFA) that contained 5 mg/mL of non-viable Mycobacterium tuberculosis H37RA (BD Biosciences) and Freund’s incomplete adjuvant (MOG35-55 was emulsified with CFA in a 1:1 volume ratio). Subsequently, 200 μg emulsified MOG35-55 was injected subcutaneously into each mouse on the posterior aspect of both ears and thigh bones on Day 0. Later, 300 ng PTX (List labs, CA, USA) in phosphate-buffered saline (PBS) was administered to each mouse via intravenous injection on Day 0 and Day 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)