Citations

Induction and assessment of EAE

Myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide was purchased from Genemed Synthesis, Inc (San Antonio, TX), was used as the immunogen. Rag1-/- mice (8-12 weeks old) received 5×106 CD4+ T cells from sex and age-matched C57BL6 mice or 1×106 CD4+ T cells from 2D2+CD24-/-MOG-/- mice [5]. The recipient mice were then immunized for the induction of EAE as we described [29]. Briefly, the T cell-recipient Rag1-/- mice were immunized subcutaneously with 200 µg MOG peptide in CFA (containing 400 µg of Mycobacterium tuberculosis) in a total volume of 100 µL. Mice were also injected with 150 ng of pertussis toxin (List Biological, Campbell, CA) in 200 µL PBS via the tail vein immediately after the immunization and again 48 hours later. EAE scores were evaluated on a scale of 0 to 5: 0, no clinical signs; 1, loss of tail tone; 2, wobbly gait; 3, hind limb paralysis; 4, moribund; and 5, death.

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)

… Cells were stimulated with ultrapure LPS from E.coli (O111:B4 serotype; List Biological Laboratories) at 10 or 100 ng/ml for up to 6 h …

• Product #421 – ULTRA PURE LPS from Escherichia coli O111:B4

EAE.
EAE was induced in female C57Bl/6J mice 8 to 12 weeks old using 150 μg of MOG35–55 (Genemed Synthesis, 110582) mixed with freshly prepared CFA (using 20 mL of incomplete Freund’s adjuvant [BD Biosciences, BD263910] mixed with 100 mg of Mycobacterium tuberculosis H-37Ra [BD Biosciences, 231141] at a ratio of 1:1 [vol/vol at a concentration of 5 mg/mL]). All mice received 2 s.c. injections of 100 μL each of the MOG/CFA mix. All mice then received a single i.p. injection of pertussis toxin (List Biological Laboratories, 180) at a concentration of 2 ng/μL in 200 μL of PBS. Mice received a second pertussis toxin injection at the same concentration 2 days after EAE induction. Mice were monitored and scored daily thereafter. EAE clinical scores were defined as follows: 0, no signs; 1, fully limp tail; 2, hind limb weakness; 3, hind limb paralysis; 4, forelimb paralysis; and 5, moribund.

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

… Pertussis toxin (List Biological Laboratories or MilliporeSigma) was dissolved in deionized water at 100 µg/ml, kept at 4 °C and freshly diluted to 2 µg/ml before use …

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)

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

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)

2.2. Cell culture
The HDFa cell line was bought from the American Type Culture Collection (ATCC; Minnesota, USAHB-8065). It was cultured in Park Memorial Institute (RPMI -1640) culture media with glutamine 2 mM (bio west, Nampa, cat n L0498-500), 10% fetal bovine serum (PAA, Pasching Austria, cat. no. A11-151) and penicillin with streptomycin 1% (Lonza, Verviers, Belgium, cat. no. DE17-602E). The HDFa cells were grown in 50 cm2 flask (Greinerbio-one GmbH Maybachstr.272636 Frickenhausen, Germany) and preserved in typical humidified incubator supplied with 5% CO2, 95% air at 37 °C (New Brunswick Scientific- Innova co-170). Cells were washed with cold phosphate buffered saline, trypsinized, harvested and centrifuged to form cell pellets. The cultured HDFa were divided into four groups as follows:

Group I (Control): The HDFa cells received no treatment.

Group II (LPS): The HDFa cells were stimulated with LPS (10 ng/mL, Escherichia coli 055:B5, List Biological Laboratories, CA, USA) for 48 h (Skioldebrand et al., 2018).

• Product #203A – LPS from Escherichia coli O55:B5

EAE Model

EAE was induced in 8-wk-old female C57BL/6 mice by a subcutaneous injection with 100 µg of myelin oligodendrocyte glycoprotein (MOG) peptide (35–55aa: MEVGWYRSPFSRVVHLYRNGK) emulsified with Complete Freund’s Adjuvant on day 0 and an intravenous injection with 400 ng of pertussis toxin (List Biological Laboratories, Campbell, CA) 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)

STAR★Methods
Key resources table

Pertussis toxin List Biological Laboratories Cat#180

Mice, EAE induction and scoring
For EAE experiments, 6–8 weeks old mice were used. mSOD1 and 5xFAD mice were maintained up to 150 days and 15 months after birth, respectively. …

Mice, aged 6-9 weeks, were immunized subcutaneously with 100 μg MOG35-55 peptide (synthesized by Toray Research Center, Japan) emulsified in complete Freund’s adjuvant containing 1 mg heat-killed Mycobacterium tuberculosis H37RA (Difco). The mice were injected intraperitoneally (i.p.) with 100 ng of pertussis toxin (List Biological Laboratories, USA) at the time of immunization and 2 days later. …

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

… PBMCs were washed and stimulated with 1 µg/ mL Staphylococcus endotoxin B (SEB; List Biological Laboratories, California, USA) in the presence of Brefeldin A (Thermo Fisher Scientific, Massachusetts, USA) at 37°C and 5% CO2 for 16 hours …