Citations

2.2. Immunization

Mice were immunized with 200 or 400 μg of AQP4 p201–220 peptide TFA salt (synthesized by Peptide Institute, Inc., Osaka, Japan) emulsified in complete Freund’s adjuvant (CFA), which was made by supplementation of Mycobacterium tuberculosis extract H37Ra (231141; BD, Franklin Lakes, NJ, USA) to incomplete Freund’s adjuvant (263910; BD). A total of 100 μL of CFA was injected intradermally at 15 sites from the base of the tail to the back on Day 0. In addition, mice received 300 or 500 ng pertussis toxin (181; List Biological Laboratories, Campbell, CA, USA) intravenously on Day 0 and intraperitoneally on Day 2. In the experiments establishing AQP4 peptide-immunized mice, we used 400 μg of AQP4 peptide; in the experiments evaluating the effects of IL-6 signal inhibition, we used 200 μg of AQP4 peptide to avoid problems such as death or skin ulcers during the experiment. Control mice were treated with complete Freund’s adjuvant and saline alone.

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

Human blood samples

For volunteers treated with endotoxin, blood samples were acquired from a random 17 out of 100 volunteers who participated in the 100LPS human endotoxemia study (NL68166.091.18, CMO: 2018-4983). …

On the day of the LPS challenge, volunteers were hospitalized at the Radboud University Medical Center and received a single intravenous administration of 1 ng/kg LPS (Escherichia coli O:113; List Biological Laboratories, Campbell, CA, USA), inducing controlled systemic inflammation. Participants were constantly monitored by a care physician for sepsis-related symptoms such as high blood pressure, high heart rate or fever, among others. …

• Product #9433 – GMP LPS Lipopolysaccharide List™ Hpt™ From Escherichia Coli Type O113

EAE-induction

EAE was induced in 8-9 weeks old C57BL/6 mice or SJL/J mice after an intradermal injection with 100µg myelin oligodendrocyte glycoprotein (MOG35-55) or proteolipid protein (PLP139-151) peptide (MD Biosciences) emulsified in CFA; final concentration of 2 mg/ml Mycobacterium tuberculosis; MD Biosciences). At days 0 and 2, 200 ng of Pertussis Toxin (PT; List Labs, Campblell, CA, USA) was administered intraperitoneally (i.p). …

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)

Tracing and surgical procedures

Rats were anesthetized with isoflurane (Isoflutek, 1000 mg/g, Karizoo, Barcelona, Spain) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) for surgery. Holes were drilled in the skull and cranial infusions of retrograde tracers were delivered into different brain regions using a Hamilton syringe (1 μl). The retrograde tracers used were FluoroGold (FG, 5-hydroxystabilamide; Cat No. 80014, Biotium, Hayward, CA, USA) and Cholera toxin-B (CTB, Cat No. 104, List Biological Laboratories Inc., Campbell, CA, USA). Injection coordinates, according to the atlas of Paxinos and Watson (2014), were (in mm) as follows: medial septum (MS) AP: + 0.6; ML: 0; DV: – 7; medial entorhinal cortex (MEnt) AP: – 6.8; ML: + 5; DV: – 8; lateral entorhinal cortex (LEnt) AP: – 6.8; ML: + 6.8; DV: – 7.6; and dentate gyrus (DG) AP: – 6.5; ML: + 6; DV: – 6.5. FG or CTB infusion volumes were 0.2 µl in all regions with an infusion rate of 0.1 µl/min and 5 min of diffusion time. After the infusion, the skin was sutured and all rats received analgesic treatment with meloxicam (Metacam, 2 mg/kg, 5 mg/ml, Boehringer Ingelheim, Barcelona, Spain). Rats remained undisturbed in their home cage for 7 days before perfusion.

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

… pertussis toxin (List labs, PTX-181) were purchased from Kasma Mall. …

Induction of EAE Model

C57BL/6J or HS mice at 6–8 weeks of age were used for the induction of EAE. The intermittent ketogenic diet was added 2 weeks in advance. The antigen emulsion was prepared by mixing an emulsion of MOG35–55 in PBS (50% w/v) with an equal volume of complete Freund’s adjuvant, supplemented with 5 mg mL−1 Mycobacterium tuberculosis. Each C57BL/6J mouse was immunized by subcutaneous injection of 0.2 mL of antigen emulsion into its back, followed by an adjuvant injection of 2 µg of pertussis toxin in 0.1 mL on the dorsum of the foot. Due to difficulty in inducing EAE in SIRPα-deficient mice, the dose was doubled in HS mice.[30] The second dose of pertussis toxin was administered 2 days after the first immunization.

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

In Vitro Assessment of Modified Manufacturing Process

Healthy human peripheral blood mononuclear cells (PBMCs) from multiple donors were obtained and cultured in fresh Roswell Park Memorial Institute (RPMI) medium with 10% fetal bovine serum in 96-well plates. Two different abicipar drug product lots were evaluated at a final concentration of 1 mg/mL: Lot 1 was used in Phase 3 studies CEDAR and SEQUOIA whereas Lot 2 was used in the MAPLE study. Bevacizumab (Avastin, Genentech; 1 mg/mL) and lipopolysaccharide (LPS; 10 µg/mL from List Biological Laboratories) were used as negative and positive controls, respectively. …

Author did not specify which List Labs LPS product was utilized in their research.
List Labs provides the following LPS products: https://www.listlabs.com/product-information/lipopolysaccharides/

Single molecule fluorescent in situ hybridization (RNAscope) and immunohistology

… Immunohistology was performed as described with minor modifications.89 In short, sections were thawed, briefly washed (PBS with 0.2% Triton X-100), and blocked (PBS with 0.2% Triton X-100 and 5% normal horse serum) for 1 hour at room temperature. The primary antibody was diluted in blocking solution and incubated for 1-2 days at room temperature. Sections were washed in PBS before being incubated with the secondary antibody diluted in blocking solution for 1 hour at room temperature. Sections were again washed in PBS and mounted with Immu-Mount (ThermoFisher). The following primary antibodies were used: goat anti-Phox2b (R&D Systems, AF4940, 1:200), rabbit anti-RFP (Rockland, 600-401-379-RTU, 1:500), chicken anti-GFP (Aves Labs, GFP-1020, 1:500), rat anti-GFP (Nacalai Tesque, GF090R, 1:1000), goat anti-CD117 (R&D Systems, AF1356, 1:400), rabbit antiTubulin b-3 (BioLegend, Poly18020, 1:1000), goat anti-CTb (List Labs, 703, 1:2000), …

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

… Control and immunised mice received 200 ng pertussis toxin (List Biological, California, US) by intraperitoneal injection on the immunisation day and 2 days post …

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 of EAE

12-week-old male hu-TIGIT KI mice were subcutaneously (s.c.) injected with 200 µg of MOG35–55 peptide (Synpeptide, Shanghai, China) and 400 µg of Mycobacterium tuberculosis in incomplete Freund’s adjuvant (Difco Laboratories, NJ, USA). Pertussis toxin (400 ng per mouse; List Biologicals, CA, USA) was intraperitoneally injected the same day and on day 2 post immunization. We also intraperitoneally administered the anti-hu-TIGIT agonistic mAb (100 μg per mouse in 100 μl PBS) or a mouse IgG1κ isotype control (clone MG1-45, BioLegend, 100 μg per mouse, in 100 μl PBS) to each mouse on days 0, 2, 4, 10, and 17. …

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)

… Diphtheria toxin (DT) List Biological Laboratories Cat#150 …

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