Citations

2.3. Antigen-induced EAE (aEAE) models

The aEAE models were established in our previous study.[4] To actively induce disease in mice, we injected a 200 mL emulsion containing 200 µg of myelin oligodendrocyte glycoprotein peptides 35–55 (MOG35–55; amino acids 35–55 of bovine MOG; Sigma-Aldrich, St. Louis, MO, USA) purified using high-performance liquid chromatography with a purity of > 95%, and Complete Freund’s adjuvant (CFA) (Sigma-Aldrich, USA) subcutaneously over six sites at the tail base and on the flank. At 0 and 24 h after immunization, mice were injected intraperitoneally with pertussis toxin (List Biological Laboratories, 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)

Inflammasome activation

Late-log cultures of Salmonella enterica serovar Typhimurium strain SL1344 (a kind gift from Dr. Brad T. Cookson, University of Washington) were grown in L-broth containing 0.3 M NaCl with shaking at 37 °C. Bacteria were washed, resuspended in PBS, and added at a multiplicity of infection of 10 bacteria per macrophage. For activation of NLRP1b, BMM from BALB/c mice were exposed to 1 µg/mL anthrax lethal toxin, prepared as a 1:1 mix of protective antigen and lethal factor (List Biological). …

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

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

… Anthrax Protective Antigen List Biological Laboratories Cat# 171E …

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Animals and EAE induction procedure

Nine-week-old male C57BL/6J mice aged (n = 14 mice/group) were obtained from Daihan Biolink Co. (Chungbuk, Republic of Korea) for use in this study. …

To induce EAE, mice (n = 14) were immunized subcutaneously on the hind flank with 1 mg/mL myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55; Koma Biotech, Seoul, Republic of Korea) emulsified in complete Freund’s adjuvant (CFA; Sigma-Aldrich, St. Louis, MO, USA) and supplemented with 5 mg/mL Mycobacterium tuberculosis H37Ra (Difco Laboratories, Franklin Lakes, NJ, USA). On days 0 and 2 post-immunization (DPI), 500 ng of pertussis toxin (List Biological Laboratories, Campbell, CA, USA) was injected intraperitoneally into the mice. Control mice (n = 14) remained non-immunized. …

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)

Cell lines and cell culture

… Both Brca2+/+ and Brca2-/- cell lines were cultured under low oxygen conditions (3% O2, 5% CO2, 37 °C) using DMEM supplemented with 10% FBS, 50 U/mL penicillin, 50 µg/mL streptomycin, 5 µg/mL insulin (Sigma-Aldrich; I0516), 5 ng/mL murine epidermal growth factor (Sigma-Aldrich; E4127), and 5 ng/mL cholera toxin (List biological laboratories; 9100B).

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

For GMP services, please fill out the following linked form: Let’s Build Something Together

KC and LSC cell culture in vitro

… Finally for CUT&RUN of LSCs, LSCs were isolated and cultured as described previously [93,94]. Briefly, corneal limbal tissue was dissected from human donor corneas, cut into small pieces, and incubated with 10 ml trypsin (0.05% trypsin—0.01% EDTA solution, Life Technologies, 25300–062) for 30 min at 37°C for 4 consecutive times. Every time, the supernatants containing the cells were collected, neutralized, centrifuged at 1,000 rpm for 5 min and cells co-cultured with a feeder layer (consisting of lethally irradiated 3T3-J2 cells) in LSC growth medium. The LSC growth medium contains 2:1 Dulbecco’s Modified Eagle Medium (Life Technologies, 21969035) and F12 (Life Technologies, 21765029), 10% Fetal Bovine Serum (Life Technologies, 10099–141), 50 mg/ml penicillin-streptomycin (Life Technologies, 15140122), 4 mM glutamine (Life Technologies, 25030081), 0.18 mM adenine grade I (Pharma Waldhof GMBH, 4010-21-2), 0.4 mg/ml hydrocortisone (Flebocortid Richter, Sanofi, AIC013986029), insulin (Humulin R, Lilly, Canada, HI0210), 2 nM triiodothyronine (Liotir, IBSA, AIC036906016), and 8.1 mg/ml cholera toxin QD (List Biological Laboratories, 9100B).

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

For GMP services, please fill out the following linked form: Let’s Build Something Together

Experimental autoimmune encephalomyelitis induction

EAE was induced by injection of 200 μg of MOG epitope MOG35-55 (RP10245, Genscript) emulsified in complete Freund’s adjuvant (CFA; 231131, BD). All mice received two subcutaneous injections into the flank and a single intraperitoneal injection of 50–100 ng pertussis toxin (PT; List Biological Laboratories, 179A). The mice received a second injection of PT on 2 d.p.i. Of note, by reducing the concentration of PT, Il12rb2fl/fl mice developed a mild disease, whereas Il12rb2del/del mice were still hypersusceptible, but within animal welfare guidelines.

• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

2.5. Immunization Procedures

Myhc-α 334–352 immunizations involved peptide/CFA emulsions containing Mycobacterium tuberculosis H37RA extract (Difco Laboratories, Detroit, MI, USA) with a final concentration of 5 mg/mL. The emulsions were administered subcutaneously (100 µg/mouse) in the inguinal and sternum regions on days 0 and 7. In some experiments, animals received emulsions in the left and right shoulders and on the right and left sides of the hip regions in split doses. Additionally, pertussis toxin (List Biological Laboratories, Campbell, CA, USA) was administered to animals 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)

… Pertussis toxin was acquired from List Biological Laboratories (#181, Campbell, Santa Clara Country, California, USA) …

2.2. EAE Induction and Clinical Scores

Mice (female, strain C57BL/6J, 18–20 g, 6–8 weeks) were immunized following a standard protocol, with minor modifications [24]. Briefly, animals were administered incomplete Freund’s adjuvant containing 4 mg/mL Mycobacterium tuberculosis (strain H37Ra) and 200 μg of the myelin oligodendrocyte protein 35–55 (MOG35–55). This was followed by intraperitoneal administration of 400 ng of Pertussis toxin on the day of immunization and two days after. …

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

EAE:

Mice were immunized subcutaneously in the flank with an emulsion containing MOG 35–55 (200μg/mouse) and CFA (200 μl/mouse; Difco Laboratories, containing M. tuberculosis H37Ra, 5 mg/ml). Pertussis toxin (200 ng/mouse; List Biological Laboratories) was administered intraperitoneally on days 0 and 2. Mice were monitored and assigned grades for clinical signs of EAE. Mice were observed for signs of EAE beginning on day 7 after 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)