Citations

… C. difficile Toxin B (TcdB) List Biological Laboratories Cat#155A …

• Product #155 – Toxin B from Clostridium difficile

… Free-floating sections were washed in 0.01 M PBS, followed by incubation in hydrogen peroxide for 20 min and incubated for 24 h at room temperature (RT) with a goat anti-CT-B antibody (1:10,000 List Biological Laboratories, CA, USA) …

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

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

IFNγ ELISPOT assay

Peripheral blood mononuclear cells (PBMCs) were collected using mononuclear cell preparation tubes (CPT) prior to treatment initiation and at 3 and 6 weeks and were cryopreserved in liquid nitrogen at 5×106 cells per vial in cryopreservation media containing 10% dimethyl sulfoxide (DMSO). The procedure for the FRα ELISPOT assay has been described previously.22 Briefly, 2.5×105 PBMCs/well were plated in triplicate into 96-well round bottom plates in 200 µL of RPMI1640 containing L-glutamine, penicillin, streptomycin, and 10% serum (T cell medium), along with cyclin D1 (control) peptide (MELLLVNKLKWNLAA), FR30, FR56, FR76, FR113, FR238, FRα protein (Sino Biological), tetanus toxoid (List Biological Laboratories), …

• Product #191 – Tetanus Toxoid from Clostridium tetani

Safety assessment in mice and RMs

For preliminary safety studies, 8-week-old female BALB/c mice were obtained from the Jackson laboratory (Bar Harbor, ME) and immunized by the intraperitoneal route with a 40-μg dose of H2O2-CampyC. In a separate control study, mice were treated with 10 μg of E. coli LPS (O111:B4, List Biological Laboratories, Campbell, CA). Animal weights were followed for up to 1 week after immunization. The second phase of our safety assessment was conducted in RM. Two adult females received an IM vaccination with the H2O2-CampyC vaccine, and two adult females received a mock, alum-only IM vaccination. Health metrics, including weight, attitude, appetite, urine, stool, temperature, and vaccine site, were monitored daily for 14 days.

• Product #201 – LPS from Escherichia coli O111:B4

BoNT administration

Research-grade, purified, native BoNT serotype A1 (BoNT-A; 150 kDa) was purchased from List Biological Laboratories (Campbell, CA, USA). It was reconstituted in 1 mg/ml phosphate buffer saline/bovine serum albumin (0.1%) to obtain a stock solution, which was aliquoted and kept frozen at −80 °C. Dilutions of aliquots were performed with 0.2% gelatin phosphate buffer (GPB) to obtain the final concentrations (see below). …

Flow cytometry and cell sorting

p63-EGFP knock-in hiPSCs were differentiated for 10 days or 6 weeks, dissociated with Accutase (Thermo Fisher Scientific) for 60–90 min at 37 °C, harvested through a 40 μm cell strainer (BD Biosciences, San Diego, CA), and collected into keratinocyte culture medium (KCM; a 3:1 mix of DMEM without glutamine and Nutrient Mixture F-12 Ham; Thermo Fisher Scientific) supplemented with 5% fetal bovine serum (Japan Bio Serum, Hiroshima, Japan), 0.4 μg/mL hydrocortisone succinate (Wako), 2 nM 3,3′,5-triiodo-l-thyronine sodium salt (MP Biomedicals, Santa Ana, CA), 1 nM cholera toxin (List Biological Laboratory, Campbell, CA), 2.25 μg/mL bovine transferrin HOLO form (Thermo Fisher Scientific), 2 mM L-glutamine, 0.5% insulin transferrin selenium (Thermo Fisher Scientific), and 1% penicillin–streptomycin. Cells were washed in phosphate-buffered saline. The 1383D2 cell line was used as an EGFP (enhanced green fluorescent protein) negative control. Cells were sorted on a SH800 Cell Sorter (Sony Biotechnology Inc., Tokyo, Japan) and the data were analyzed using SH800 software.

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

Preparations

… The preparation of the keratinocyte culture medium (KCM) is as follows; Dulbecco’s modified Eagle’s medium (DMEM; Sigma aldrich, MO, USA) and F-12 medium (Sigma aldrich) were mixed at a 3:1 ratio. The medium was supplemented with 3.3 mM l-glutamine solution (Sigma Aldrich), 84 μM gentamicin (Merck Sharp and Dohme, NJ, USA), 0.3 μM amphotericin B (Bristol-Myers Squibb, NY, USA), 0.25 μM hydrocortisone (Kowa, Nagoya, Japan), 140 mU/mL insulin (Lilly, IN, USA), 2.0 nM 3,3′,5-Triiodo-l-thyronine (MP Biomedicals, CA, USA), 1.0 nM cholera toxin (List Biological Laboratories, CA, USA), …

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

Cell culture and stimulation

CA-Mφ were generated by priming BMM with 150 U/ml IFN-γ (R&D Systems, Minneapolis, MN) for 6 h, and then stimulating cells overnight with 150 U/ml IFN-γ and 100 ng/ml LPS (from Salmonella typhimurium; no. 225; List Biological Laboratories, Campbell, CA). …

• Product #225 – LPS from Salmonella typhimurium

Cell lines

… MCF-10A cell lines were maintained in the full growth medium, which consisted of DMEM/F12 (1:1) (Cat#11330-032, Gibco) supplemented with 5% horse serum (Cat#16050-122,
Invitrogen), 10 mg/ml insulin (Cat#12878-44, Nacalai Tesque), 0.5 mg/ml hydrocortisone (Cat#1H-0888, Invitrogen), 100 ng/ml cholera toxin (Cat#101B, List Biological Laboratories), …

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