Citations

2.4.4 Purification of PA and LF

PA83 was expressed and purified by the Barth group. After purification, PA83 was activated in vitro to PA63 and stored at -80 °C.
LF was purchased from List Biological Laboratories (see 2.1.7) and stored at -80 °C.

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

2.1.2 Study II: Examination of the contribution of neuroinflammatory processes such as
BBB leakage and focal inflammation to changes in mechanical properties

For in vivo MRE and MRI experiments adoptive transfer EAE was induced in 21 SJL mice (female, 9-12 weeks, Janvier, SAS, France, registration number G106/19) by the transfer of myelin proteolipid protein (PLP)-reactive lymphocytes. SJL mice were used as the animal strain of choice to induce EAE (active and adoptive transfer), as they display a high disease susceptibility after induction. PLP-reactive lymphocytes were obtained from 22 donor mice, which were actively immunized as previously described (31, 34) using 200 mg of the myelin peptide PLP139-151 emulsified with 200 ml complete Freund´s adjuvant (Thermo Fischer Scientific, United States) and 800 mg Mycobacterium tuberculosis
H37Ra (Difico, United States). As described in (31), on days 0 and 2 postimmunization (p.i.), 250 ng pertussis toxin (List Biological Laboratories, United States) was injected intraperitoneally. Donor animals were sacrificed on day 10 p.i., axillary and inguinal lymph nodes were collected and cells cultured as described (35). Cells were harvested after 4 days in culture with RPMI 1640 medium (supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin and 10 % fetal bovine serum) (Gibco, Thermo Fischer Scientific) containing 12.5 mg/ml PLP. Into each of the 21 recipient mice 30 million cells were intraperitoneally injected. …

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)

2.2.1 Active EAE

For Studies I, II and III active EAE was induced in SJL mice. Therefore, 9 – 12-week-old mice (Janvier, SAS, France) were subcutaneously immunized with an emulsion containing 250 µg of a PLP139-151, 800 µg M. tuberculosis H37Ra (Difco, USA) and 100 µl of complete Freund’s adjuvant (CFA; Thermo Fisher Scientific, USA) in addition to an intraperitoneal injection of 250 ng pertussis toxin (List Biological Laboratories, USA) on the same day and 2 days later. …

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

… PTX was from List Biological Laboratories, Inc. (Campbell, CA); …

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)

… Lipopolysaccharide (LPS) from Salmonella typhimurium was obtained from List Biological Laboratories (Campbell, CA) …

• Product #225 – LPS from Salmonella typhimurium

Alveolar macrophage Ex Vivo stimulation

AMs were isolated by bronchoalveolar lavage and pooled from 5 mice per group. Cells were plated at a density of 5 x 104 cells/well (96-well plate) in complete RPMI (RPMI plus FBS (10%, VWR), L-glutamine (2mM, Invitrogen), and Penicillin-Streptomycin (100 U/ml; Invitrogen) and allowed to adhere overnight in a 37°C humidified incubator (5% CO2). Media with antibiotics and non-adherent cells were washed out prior to stimulation. AM were stimulated with LPS (LPS from Salmonella Minnesota, List Biologicals, #R595, 10 ng/ml), Pam3Cys (Pam3CSK4, EMC Microcollections, GmbH, 10 ng/ml), or H37Rv (effective MOI ~2:1). H37Rv was prepared by culturing from frozen stock in 7H9 media at 37°C for 48 hours to O.D. of 0.1–0.3. …

• Product #304 – LPS from Salmonella minnesota R595 (Re)

… mice were injected with PTX (0.25 µg, List Biological Laboratories, Campbell, CA, USA) on D0 and D2 [53,54]. Clinical score was evaluated in a blind method by fundus photographs …

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 was purchased from List Biological Labs Inc. (Campbell, CA, 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)

Immunization.

Mice were immunized weekly for 6 weeks by oral gavage with 5 mg of ground blanched peanut (Western Mixers Produce & Nuts) or 1 mg of NP-Ovalbumin (LGC Biosearch) with 10 µg CT (List Biologicals, Lot: #10167A2 and #10165A1) in 200 µl of 0.2 M sodium bicarbonate buffer per mouse based on a protocol modified from (10). …

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

EAE]

EAE was induced in 7- to 13-wk old female mice as described previously.81 Briefly, mice were immunized subcutaneously with 150 μg MOG35-55 (MEVGWYRSPFSRVVHLYRNGK, EZBiolab) in PBS and complete Freund’s adjuvant (BD Biosciences, cat # 263910) containing 4 mg/mL M. Tuberculosis H37Ra (BD Biosciences, cat # 231141) with or without intraperitoneal injection of 250 ng pertussis toxin (List Biological Laboratories, cat # 180) on day 0 and day 2. …

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