Citations

Colony-forming assay

Colony-forming assay (CFA) was performed as previously reported (Sasamoto et al., 2020). Briefly, limbal epithelial cells were seeded on the mitomycin C (MMC) (MilliporeSigma)-treated 3T3-J2 feeder cell layer at 500 cells per well on 6-well plates. The cells were cultured in keratinocyte culture medium (KCM) supplemented with 10 ng/mL KGF and 10 μM Y-27632. KCM was composed of DMEM without glutamine and Ham’s F-12 Nutrient Mix (Thermo Fisher Scientific) combined at 3:1 ratio, supplemented with 10% FBS, 0.4μg/ml hydrocortisone hydrogen succinate (MilliporeSigma), 2nM 3,3′,5-triiodo-l-thyronine sodium salt (MilliporeSigma), 1 nM cholera toxin (List Biological Laboratories, Campbell, CA), …

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

Experimental Human Endotoxemia (IV LPS Administration)

All experimental endotoxemia-related procedures were performed as described previously and were identical on both LPS challenge days (days 0 and 7) [19, 22]. In short, subjects were admitted to the research unit of the Radboud University Medical Center for 8 h. Subjects had to refrain from alcohol and caffeine (24 h) and food and drinks (12 h) prior to LPS administration. A radial artery catheter (BD Infusion Therapy Systems, Sandy, UT, USA) and antebrachial venous cannula were placed to allow serial blood sampling, hemodynamic monitoring, and administration of fluids and LPS, respectively. In the 45 min prior to LPS administration, hydration fluids (2.5% glucose/0.45% sodium chloride) were administered as a 1.5-L prehydration bolus to reduce the risk of vasovagal collapse [23] and thereafter at a rate of 150 mL/h for the remainder of the experiment. Directly after prehydration, a bodyweight-adjusted bolus dose of 1 ng/kg LPS (Escherichia coli-derived, Type O113, lot No. 94332B1; List Biological Laboratories, Campbell, CA, USA) was administered. Blood samples were serially obtained to construct time-concentration curves of circulating cytokines.

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

… Bordetella Pertussis toxin List Biological Labs Cat. #: 180 …

MOG35-55-induced EAE
EAE was induced in mice using MOG35-55 as described by Bittner et al. (2014). Briefly, 8-week-old endothelial-specific Arf6 knockout mice (Arf6f/f; Cdh5-CreERT2) and their control sibling littermates or C57BL/6J mice were immunized with MOG35-55 in CFA emulsion followed by administration of pertussis toxin (PTX) in PBS at day 0 and again at day 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)

2.7.4 Tracer intramuscular injection

Adult female Lister Hooded rats (220–290 g; Charles River; husbandry conditions as above), either naïve (n = 5) or 10 weeks after SCI (spinal C7 bilateral contusion, as above; n = 3) had the right biceps brachii intramuscularly injected with CTB. Rats were anesthetized with isoflurane (5% induction and 2.5% maintenance in 1 L/min O2). Analgesia (carprofen, CarprieveTM, 5 mg/kg) was administered subcutaneously, and the right forelimb was shaved and disinfected with 4% w/v chlorhexidine gluconate. The skin was incised and retracted, and the fascia was carefully opened for clear visualization of the two heads of the muscle. A total of 12 μl of 0.5% CTB diluted in distilled water (104 Cholera Toxin B Subunit; #10433A1, List Biological Laboratories, Inc.) was injected into the right biceps using 10 μl Hamilton syringes (#80300, 26s-gauge, Hamilton). The short head of the biceps received 8 μl of CTB, divided into two deep injections of 2 μl and two superficial injections of 1 μl aiming the motor endplates (Tosolini & Morris, 2012) and two deep injections of 1 μl aiming at the region halfway between the proximal end of the muscle and the motor endplates (Figure 6a). The long head of the biceps received 4 μl of CTB, divided into one deep injection of 2 μl and one superficial injection of 1 μl aiming the motor endplates, and one deep injection of 1 μl aiming the region halfway between the proximal end of the muscle and the motor endplates (Figure 6a). The muscle was wiped before and after injections with a cotton bud. The needle was inserted parallel to the muscle fibers, the tracer was injected slowly, and the needle was kept in place after injection for at least 30 s. After injections, the skin was sutured, and saline administered subcutaneously. Rats recovered from anesthesia in a heated recovery chamber.

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

Surgical procedures

Mice were anesthetized with an intraperitoneal injection of the ketamine and xylazine mixture (100 mg/kg and 10mg/kg, respectively). For the creation of SNI, muscles were displaced to expose the right sciatic nerve, and the nerve was ligated proximal to its trifurcation. The sciatic nerve was completely transected below the ligation site with fine surgical scissors. Rats were anesthetized with an intraperitoneal injection of the ketamine and xylazine mixture (90 mg/kg and 8mg/kg, respectively). SNI in rats was performed following the same procedures as for mice. To create a dorsal column lesion in the spinal cord, a dorsal laminectomy was performed at the T9 level and bilateral dorsal columns with adjacent lateral columns were cut out with iridectomy scissors. To visualize regenerating axons, cholera toxin subunit B (CTB; List Biological Laboratories) was injected using a protocol modified slightly from that in the previous report 21. Briefly, after the sciatic nerve between the thigh muscles was exposed, a small incision was made on the perineurium just proximal to the trifurcation site. Two microliters of unconjugated CTB solution (1% in PBS) were slowly injected using the Hamilton syringe through the perineural incision and animals were killed 5 d after the injection.

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

Misoprostol and LPS administration

For each treatment arm, manure was manually evacuated prior to administration of misoprostol (M-PO, M-PR) or water (CON). For per os administration (M-PO), misoprostol tablets (100 μg; Lupin Pharmaceuticals) were dissolved in 30 mL of water and administered via oral syringe. This was followed by administration of 30 mL of water through the same syringe for a total volume of 60 mL delivered. Water (60 mL) was also administered per rectum via 16-inch 8-French red-rubber catheter advanced approximately 30 cm into the rectum. For per rectum administration (M-PR), misoprostol tablets dissolved in 30 mL of water were infused via red-rubber catheter as described above, followed by infusion of 30 mL of water through the same syringe for a total volume of 60 mL delivered. Water (60 mL) was then administered orally via syringe. Horses in the CON treatment received water per os (60 mL) and per rectum (60 mL) as already described. Immediately following treatment administration (M-PR/M-PO/CON), LPS (10 µg/mL; List Biological Labs) was administered IV at 30 ng/kg diluted in 500 mL saline (0.9% NaCl) solution as a 30-minute continuous rate infusion.

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

Optic nerve crush and intraocular injections

… All viruses were injected in a volume of 3 µl and a titer of 1 × 1013 gc/ml 2 weeks prior to optic nerve crush to insure adequate time for gene deletion or transgene expression at the time of nerve damage. Two days prior to the end of a 14-day survival period, cholera toxin B subunit (CTB, 3 µl/eye, 2 µg/µl, List Biological Laboratories, Inc., 103B) was injected intraocularly as an anterograde tracer to label axons regenerating through the optic nerve. …

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

Maintenance culture conditions

All MCF10A-derived cell lines were maintained in standard MCF10A growth medium without antibiotics (DMEM/F12 medium (Wisent Bioproducts, 319-075-CL), 5% horse serum (Gemini Bio, 100508), 10 µg ml−1 insulin (Thermo Fisher Scientific/Gibco, A11382II), 0.5 mg ml−1 hydrocortisone (Sigma-Aldrich, H4001), 20 ng ml−1 EGF (R&D Systems, 236-EG-01M) and 100 ng ml−1 cholera toxin (List Biological Laboratories, 100B)). …

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

EAE and EAE tissue isolation

Female C57BL/6 mice (8–10 weeks old) from Jackson Laboratories were immunized subcutaneously with MOG 35–55 peptide (50 μg/100 μl, Protein and Nucleic acid facility, Stanford University School of Medicine, Stanford, CA) emulsified in complete Freund’s adjuvant (CFA) (Thermo Fisher Scientific) containing 10 mg/ml of heat inactivated Mycobacterium tuberculosis H37RA (Sigma-Aldrich). 50 μl emulsion was injected at one site into each hind flank. On the day of immunization and 48 h later, animals received intraperitoneal injections of 300 ng of pertussis toxin (List Biological Laboratories). …

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)

… The assay mixtures were allowed to incubate at rt for 30 min, before the addition of 25 µL of SNAPtide substrate flP6 (List Laboratories) to initiate the enzyme reaction, …

• Product #523 – SNAPtide® Peptide Substrate flP6(DABCYL/5-IAF) for C. botulinum Type A Neurotoxin