Citations

EAE induction in mice

… Eight-ten week-old C57BL/6 N female mice (Charles River Laboratories, Montreal, QC, Canada), housed under specific pathogen free conditions, 4/cage, on a 12:12 h light cycle were used in this study. EAE was induced by subcutaneous immunization with 100 μg myelin oligodendrocyte glycoprotein (MOG35–55) in emulsion of a 1:1 volume with complete Freund’s adjuvant (CFA, containing 4 mg/ml of heat killed Mycobacterium tuberculosis, Difco Laboratories, Detroit, MI, USA). The mice were also injected with intravenous Bordetella pertussis toxin (200 ng; PTX, List Biological Laboratories Inc., Campbell, California, USA) in 1X Phosphate Buffered Saline (PBS) at the time of, and two days following, 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)

Detection of PA and Anti-PA IgG in Serum

PA was measured in serum using PA-ECL screening assay kit MesoScale Discovery (L15CA-1), which utilizes the anti-PA antibody. To quantitate the levels of PA in each serum sample, a standard curve (0–100 ng/mL) was analyzed in parallel on each assay day by using recombinant PA (List Biological Laboratories, Inc., 171E). Test samples were assayed in duplicate.

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

2.1. RR-EAE Induction

SJL/JCrl female mice were purchased from Charles River (Calco, Italy). All mice were housed in pathogen-free conditions with access to food and water ad libitum. RR-EAE was induced in female wild type mice (7 weeks of age, weighing 18.5 ± 1.5 g) via a subcutaneous injection at two different sites in the right and left flanks with an emulsion (200 μL total) containing 200 μg of proteolipid protein peptides spanning amino acids 139–151 (PLP) (Espikem, Florence, Italy) in incomplete Freund’s adjuvant (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) supplemented with 600 μg Mycobacterium tuberculosis (strain H37RA; Difco). Mice were injected in the tail vein with 400 ng pertussis toxin (List Biological, Campbell, CA, USA) in 100 μL of phosphate buffer saline solution (PBS, pH 7.6) immediately and 48 h after the 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)

EAE induction

For active EAE induction, age- and sex-matched mice were immunized s.c. with MOG35-55 (Genscript, purity >98%) peptide (300 μg) emulsified in CFA containing 4 mg/ml heat-killed MTB (Cat#7001,Chondrex). 200 ng Pertussis toxin (Cat#181239A1, List Biological Laboratories) dissolved in PBS was administered i.p. on days 0 and 2. …

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

EAON Model

EAON was induced in an experimental autoimmune encephalomyelitis (EAE) mouse model (WT, n = 12; limitrin KO, n = 18), as previously described.4,5,23,24 Areas draining into axillary and inguinal lymph nodes of 7- to 8-week-old mice were subcutaneously injected with 200 µg myelin oligodendrocyte glycoprotein (MOG35–55 peptide fragment MEVGWYRSPFSRVVHLYRNGK; GL Biochem Ltd., Shanghai, China) emulsified in 100 µL solution containing 50% complete Freund’s adjuvant and 10 mg/mL heat-killed H37Ra strain of Mycobacterium tuberculosis (Difco Laboratories, Detroit, MI). Pertussis toxin (List Biological Laboratories, Campbell, CA) dissolved in PBS (200 ng/mouse) was administered intraperitoneally on days of immunization and again 48 hours later. …

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 Optic Nerve Transection and NSC Transplant

… Three days before tissue collection, animals received intravitreal injections of cholera toxin subunit B (CTB; 2 µL, 1 µg/µL; List Biological Labs, Campbell, CA, USA) to anterogradely label RGC axons. Animals were perfused transcardially with cold PBS, followed by 4% PFA, and post fixed in 4% PFA overnight. The eyes were dissected and stored in PBS. The optic nerves were dissected and cryoprotected in 30% sucrose.

Retina and Optic Nerve Immunohistochemistry

… Primary antibodies to CTB (1:1000; List Biological Labs), …

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt
• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

Immunization

Immunization was performed as described previously (17). Briefly, on days 1, 7, and 17, the mice were intranasally immunized with 5 μg of ovalbumin (OVA) (Sigma-Aldrich) alone or 10 μg of Alcaligenes LPS or 1 μg of cholera toxin (CT) isolated from Vibrio cholerae (List Biological Laboratories, Campbell, CA, USA) in 15 µL of PBS and administered as 7.5 µL in each nostril of mice without anesthesia. One week after the final immunization, nasal wash, bronchoalveolar lavage fluid (BALF), serum, nasal passage, NALT, cervical lymphoid nodes (CLNs), and spleen were collected as previously described (17, 18) and used for analysis.

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

EAE induction and Treg-specific deletion of Piezo1

Active EAE was induced in mice by immunization with MOG35–55 peptide (1 mg/ml) (MEVGWYRSPFSRVVHLYRNGK) emulsified in CFA supplemented with heat-killed Mycobacterium tuberculosis H37Ra (5 mg/ml). One hundred microliters of emulsion (EK-0111, Hooke Laboratories, Lawrence, MA) was injected subcutaneously at two sites over the flank region. In addition, 150 ng of pertussis toxin (PTX) (List Biological Laboratories, Campbell, CA) was injected intraperitoneally on the day of immunization and 48 hours later, as described previously (24, 56). …

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)

2.13. Recall antigen assay

Approximately 1 × 105 PBMCs per well were seeded in a 96-well tissue culture plate and cultured in 100 μl of RPMI-1640 supplemented with 10% FBS overnight at 37 °C and 5% CO2. havPD-1 EVs in various concentration were added to the cells at 50 μl per well. In control group, 50 μl of Atezolizumab or isotype control IgG1 were added. After 1-h incubation, 50 μl of 0.8 μg/ml Tetanus Toxoid (List labs, 191A) was added to wells. After 5-day culture, supernatant was collected and IFNγ was measured (R&D Systems, DIF50) according to manufacturer’s instructions.

• Product #191 – Tetanus Toxoid from Clostridium tetani

For in vitro class-switching assays, cells were stimulated with 20 ng/mL interleukin (IL)-4 (R&D Systems, Minneapolis, MN, USA) and 10 µg/mL lipopolysaccharide (LPS; List Biological Laboratories, Campbell, CA, USA) for the time periods described …

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/