Citations

Human Donor SMILE Lenticule Acquisition and Preparation

Fresh lenticules (left and right-eye; n = 113) were obtained from consenting patients (both female and male; age range: 21–50 years) that elected to undergo SMILE laser refractive surgery at the Envision Eye Centre (Sydney, Australia). Lenticules were collected and kept in sterile saline at 4 °C until experimentation. They were conditioned to receive CLECs by submerging them in dKSFM (Thermo Fisher, Waltham, MA) comprising 10 ng mL−1 epidermal growth factor (R&D Systems, Minneapolis, MN), 100 ng mL−1 cholera toxin (List Biological Laboratories, Campbell, CA), and 100 U mL−1 penicillin–streptomycin (Thermo Fisher) for 2 weeks at 37 °C in 5% CO2, otherwise referred to as complete media. Lenticules were used either immediately after conditioning or following gamma (ɣ)-irradiation with 1000cGy for 20 min. Next, they were washed twice in PBS and further incubated in complete dKSFM media without FBS for 3 days prior to receiving a suspension of cells or tissue explants for subsequent cultivation. This strategy was employed to mitotically inactivate and/or deplete viable lenticule-associated keratocytes.

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

Materials and Methods

Animals.
Mice of both sexes were used between 4-17 weeks of age and maintained on a C57BL/6
background under specific pathogen-free (SPF) conditions in Allentown caging with aspen shavings and
LabDiet5010 chow (Purina, St. Louis, MO) and RO water. TNFDARE/+ mice8 were rederived by the Genome
Editing Shared Resource, Rutgers Cancer Institute of New Jersey and subsequently crossed to TcrdGDL
mice67. TNF+/+ littermates were used as controls. For suppression assays, wildtype C57BL/6 mice or
TcrdEGFP68 mice were used between 8-14 weeks of age. To assess IEL proliferation, 200 µg of
5-ethynyl2′-deoxyuridine (EdU)(Sigma-Aldrich) was administered i.p. daily for 4 days. To induce gd T cell depletion
in 4-week-old TcrdGDL; TNFDARE/+ mice, 15 ng of diphtheria toxin (List Biological, Campbell, CA) or
endotoxin-free PBS per gram body weight was administered i.p. 24 and 48 h prior to the experiment. …

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

… Cholera toxin (AZIDE-FREE) from Vibrio cholerae List Biological Laboratories 100B …

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

EAE Induction

Female C57BL/6J mice at 6–8 weeks old were purchased from Experimental Animal Center of Air Force Military Medical University. EAE was induced with 200 ug MOG35 − 55 peptide (NJPeptide, China) emulsified in incomplete Freund’s adjuvant (Sigma – Aldrich, USA) added with 5 mg/mL Mycobacterium Tuberculosis H37RA (Difco, USA). Each mouse was immunized with the emulsion subcutaneously into four sites at the back followed by intraperitoneal injection of 200 ng pertussis toxin (List Biological Laboratory). …

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)

…A significant part of CRM197 in the world is produced by cultivation of the non-toxic strain C. diphtheriae C7(β197) [11]. This method is used by List Biological Labs (USA). Advantages of this method include correct folding of the target protein and its secretion into the culture medium, which facilitates further purification of the product. …

• Product #149A – Diphtheria Toxin CRM197, Mutant

Experimental Autoimmune Uveitis Model Establishment

We prepared an emulsion by adding Mycobacterium tuberculosis toxin (Difco Laboratories, Franklin Lakes, NJ, USA) to complete Freund’s adjuvant (Difco Laboratories) to achieve a concentration of 2.5 mg/mL, which was then mixed with an equal volume of interphotoreceptor retinoid-binding protein (1–20) (IRBP1–20; 2 mg/mL; GiL Biochem, Shanghai, China) PBS solution. Subsequently, 200 µL of the emulsion was subcutaneously injected into anesthetized mice for immunization. In addition, a 0.25-µg intraperitoneal injection of pertussis toxin (List Biological Laboratories, Campbell, CA, USA) was administered on the day of immunization and the following day. …

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)

In vivo cell depletions

Animals were depleted of CD4+ or CD8+ T cells as previously described [19,85]. Briefly, animals were treated with 0.5 mg of rat anti-CD4 mAb GK1.5 (Leinco) or 0.2 mg rat anti-CD8 mAb 2.43 (Leinco) in 200 μl of DPBS i.p. one day prior to challenge. Treatment was repeated once weekly for the entirety of each study. For depletion of B cells, animals were treated with 250 μg of rat anti-CD20 mAB MB20-11 (BioXcell) in 200μl of DPBS i.p. one week prior to challenge [86]. Anti-CD20 treatment was repeated on day 14 post infection. Control animals for both T and B cell depletions received equivalent dosages of rat or mouse IgG (Leinco or Southern Biotech) respectively. Upon euthanasia, depletion of splenic CD4+ T, CD8+ T, or B cell populations were confirmed to be ≥ 90% effective via flow cytometry. TReg were depleted in DEREG animals by administering 1 μg DTX (List Biological Laboratories) resuspended in 100 μL DPBS i.p. to each animal on day 14 and 15 post challenge [59]. WT animals were administered an identical dose of DTX as a control. Blood was collected from animals to confirm systemic depletion of FoxP3+CD4+ T cells seven days post treatment (S3A Fig).

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

2.2. EAE induction and treatment
EAE was induced according to previously established methods [17,18]. Briefly, mice were subcutaneously immunized with 200 mg MOG 35–55 peptide emulsified in complete Freund’s adjuvant containing 0.4 mg Mycobacterium tuberculosis H37 Ra (BD Difco) and intraperitoneally injected with 200 ng pertussis toxin (List Biological Laboratories) on days 0 and 2 post-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)

Tracer injection

Rats received a subcutaneous injection of the nonsteroidal anti-inflammatory ketoprofen
(2.5mg/kg, s.c.). and were anesthetized with isoflurane (5% induction, 2.5% maintenance) in
O2 before being placed in a stereotaxic frame. Animals were placed on a heating pad for the
entire procedure. Lidocaine (1mg/kg) was injected locally before incision of the skin. Then,
craniotomies were performed above the brain areas of interest. A retrograde tracer cholera
toxin subunit B (CTB, 0.5 μl, List Labs, Campbell, CA, USA) was injected into the right NAcC
[anteroposterior (AP), +1.1mm for males and +1.2mm for females from bregma; lateral (L),
2.2mm from the midline; dorsal (D), -7.4mm from the skull surface]. Following injection, the
incision was sutured, and the rats were placed in their homecage for 2 weeks before brain
collection.

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

EAE

EAE was induced in 8–12 weeks old female C57BL/6J mice as previously described (17, 70). … On day 0, mice were immunized by injecting 50 μl of the emulsion subcutaneously (SC) into each of two sites on the lateral abdomen. On days 0 and 2, the mice were injected IP with 250 ng pertussis toxin (List Biologicals, catalog no. 181) dissolved in PBS. On day 7, they were weighed and began scoring.

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