Citations

2.9. Immunization

Immunization involved Myhc-α 334–352 emulsified in complete Freund’s adjuvant (CFA) supplemented with an extract of Mycobacterium tuberculosis H37RA (Difco Laboratories, Detroit, MI, USA) to 5 mg/mL concentration [18]. The peptide emulsion was subcutaneously injected (100 µg/mouse) into the shoulder and hip regions (left and right) in split doses. In addition, animals received pertussis toxin (List Biological Laboratories, Campbell, CA, USA), intraperitoneally (100 ng/mouse) on days 0 and 2 after the first immunization.

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)

Experiment 2: Cholera toxin B microinjection

Rats received 100 nl of the retrograde tracer Cholera Toxin B subunit (CTB; 1% w/v in ACSF, List Biological Laboratories, Campbell, CA) over 5 min into the PFA three weeks prior to euthanasia as previously described [41].

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

4.1. Cell Culture and Generation of Human TEC Culture

… Cells were cultured in a humidified atmosphere of 5% CO2 in a medium composed of DMEM:Ham’s F-12 (3:1 ratio), 10% fetal calf serum (FCS) (ThermoFisher, Waltham, MA, USA), adenine (0.18 mM, Sigma-Aldrich, St. Louis, MO, USA), hydrocortisone (0.4 μg/mL, Sigma-Aldrich, St. Louis, MO, USA), insulin (5 μg/mL, Eli Lilly, Indianapolis, Indiana), cholera toxin (0.1 nM, List Labs, Campbell, CA, USA), glutamine (4 mM, ThermoFisher, Waltham, MA, USA), triiodothyronine (2 nM, Sigma), and antibiotics. Epidermal growth factor (10 ng/mL, Austral Biologicals, San Ramon, CA, USA) was added to the medium after 48 h of culture. …

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

… Recombinant Corynebacterium diphtheriae toxin was obtained from Fisher (List Biological Laboratories, Cat. #150) …

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

Diet and treatment

Breeding cages were randomly assigned to one of the experimental isocaloric diets (AIN93G, BioServ): standard (Stand, BioServ #S7628), n-3 long chain (LC)-PUFA enriched (n-3, BioServ #S7630; soybean oil replaced with Menhaden Fish oil) or n-6 LC-PUFA supplemented (n-6, BioServ #S7629; based on corn oil). The detailed composition of the diets is presented in our previous publication (Chumak et al., 2022). The day of birth was defined as postnatal day (P) 0. At P9 pups received a single intraperitoneal injection of lipopolysaccharide (LPS from E. coli O55:B5, 1mg/kg, Cat# 423, List labs) or equivalent volume of saline. The dams remained on the experimental diet until offspring sacrifice (P10-P12). Efforts were taken to include equal number of pups of both sexes in each experimental group.

• Product #423 – ULTRA PURE LPS from Escherichia coli O55:B5

Diphtheria toxin (DT) injection

Mice received 500 ng diphtheria toxin (DT, Cat# 150, List Biological Laboratory) in 100 µL distilled, 486 deionized water (ddH2O) through intraperitoneal injection (i.p.) (Binnewies et al., 2019) on 31, 32 487 and 34 dpi (days post-injury), …

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

… 2) Diphtheria Toxin: DTx (List Labs, cat# 150) was suspended in filter sterilized phosphate buffered saline (PBS) to create stock aliquots of 2 mg/mL solution, stored at -80 °C. …

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

… The following antigens were used: … tetanus toxin (TTx) (List Biological Laboratories, Campbell, USA), …

• Product #190 – Tetanus Toxin from Clostridium tetani

Induction and evaluation of active EAE

For the construction of chimeric mice, Rag2-/- mice were adoptively transferred with 107 wild-type (Wt) T cells, Mid1-/- T cells, or a mixture of CD45.1-expressing Wt and CD45.2-expressing Mid1-/- T cells at a 1:1 ratio. Wt, Mid1-/-, or chimeric mice were then used for the induction of active EAE as previously reported 25. Briefly, mice were subcutaneously injected with a 200 μL emulsion that contains 1 mg/ml MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK), 2.5 mg/ml Mycobacteria tuberculosis H37Ra (B.D.), and 100 μL Freund’s adjuvant (Sigma). On the day of immunization and two days after immunization, mice were intraperitoneally injected with 200 ng of pertussis toxin (List Biological Laboratories Inc.).

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)

Processing of material for light microscopy.

… Subsequently, for fluorescence microscopy, slices were stained for GAD67 with mouse
anti-GAD67 (MAB5406; 1:2,000, Millipore), for VGLuT2 with guinea-pig anti-VGLuT2 (AB2251-I; 1:2,000, Millipore), for
CTB with goat anti-CTB (703; 1:2,000, List Labs), …

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