Citations

2.4 Peripheral blood mononuclear cells (PBMCs) functional assay; in vitro stimulation and cytokine measurement

Out of 99 healthy individuals, blood samples from 61 individuals were available for the in vitro functional assay. Whole blood samples were prepared for PBMCs extraction facilitated by Ficoll-Paque PLUS density gradient centrifugation. Cleaned PBMCs were suspended in PBS with EDTA, and cells were counted using Bio-Rad TC20 Automated Cell Counter (Bio-Rad, CA, US). 3 × 106 cells were used for stimulation. PBMCs were challenged with or without 8.33 ng/ml of lipopolysaccharide (LPS, List Biological Laboratories Inc.) for 4 h at 37°C, centrifuged at 400 g and supernatants stored at −80°C until further analysis. All incubations were performed in triplicates and thereafter pooled. The pooled samples were analysed in singlicates using the Bio-Plex Pro Human Cytokine Grp 1 Panel 17-plex assay (Cat. #M5000031YV, Bio Rad Laboratories, Inc. Hercules, USA, including G-CSF, GM-CSF, IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p70), IL-13, IL-17A, MCP-1, MIP-1β, TNFα) according to the manufacturer’s protocol.

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/

… MCF-10A human breast normal cells were cultured in DMEM/F-12 (Welgene) containing 100 ng mL−1 Cholera Toxin (List Biological Laboratories) and 10% Human Keratinocyte Growth Supplement (HKGS, Gibco) …

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

STAR★Methods
Key resources table

Goat anti-CTb antibody List labs 703, RRID: AB_10013220

Inhibitory neuron (IN) marker expression in CTb tagged PFC LRG neurons

5-7 days after CTb injection, mice were transcardially perfused with PBS followed by 4% PFA solution, and brains were post-fixed for at least one day. Coronal sections (75 μm) were obtained using a vibratome, and immuhistochemistry was performed (as described above). The following primary antibodies were used to stain for IN markers: rabbit anti-PV (Swant; diluted 1:200); rat anti-SST (Millipore, diluted 1:200); rabbit anti-VIP (Immunostar, diluted 1:200); rabbit anti-NPY (Immunostar, diluted 1:500); rabbit anti-calretinin (Immunostar, diluted 1:500); rabbit anti-nNOS (Life technologies, diluted 1:500), and goat anti-CTb (List, diluted 1:500). …

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

… A standard curve was generated using wells that received purified C. difficile toxin A (List Biological Labs) …

• Product #152C – Toxin A from Clostridium difficile

2.6. ELISA for CTB- and LTB- specific human Abs and rice-specific IgE

CTB– or LTB-specific antibody responses were determined by using an electrochemiluminescence-based ELISA (MSD, Kenilworth, NJ, USA) and a surrogate standard for CTB-specific human IgG and IgA. In brief, the wells of 96-well microtiter plates (multi-array 96-well plates, MSD) were each coated with 30 µL CTB (List Biological Laboratories, Campbell, CA, USA) …

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

… cholera toxin (List labs, #100B), …

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

Mouse Model of EAE and Study Design

Animal experiments were conducted in accordance with national and institutional guidelines for the care and use of laboratory animals and with directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 and were approved by the Berlin State Office for Health and Social Affairs (LAGeSo, registration number G106/19). Experimental autoimmune encephalomyelitis was actively induced in 9- to 12-week-old SJL/J female mice (Janvier Labs, France; n = 29) by subcutaneous immunization with 250 μg proteolipid protein peptide (PLP139–151; purity 95%; Pepceuticals, Leicester, United Kingdom) and 800 μg Mycobacterium tuberculosis H37Ra (Difco, Franklin Lakes, NJ) diluted in 100 μL complete Freund’s adjuvant. In addition, 250 ng pertussis toxin (List Biological Laboratories, Campbell, CA) were injected intraperitoneally on day 0 (day of immunization) and day 2 of the study. …

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 and scoring.

Mice were immunized with CFA/MOG emulsion in the lower back on day 0. The emulsion was prepared by mixing MOG35-55 peptide (United Biosystems, catalog U104628) and CFA (MilliporeSigma, catalog F5881) with additional M. tuberculosis (BD Difco, catalog 231141; 200 μg/mouse). The mice also received an intraperitoneal injection of Pertussis toxin (PTx) (200 ng/mouse) (List Biological Technologies, catalog 180) on days 0 and 2. Unless otherwise noted, we induced EAE with this method, as Type-A EAE (100 μg MOG35-55/mouse and 200 μg M. tuberculosis/mouse) (10). In some experiments, Type-B EAE (10) was induced with CFA/MOG injection on days 0 and 7 (100 μg MOG35-55/mouse, 400 μg M. tuberculosis/mouse) and PTx (200 ng/mouse) on days 0, 2, and 7. EAE was scored as previously described (2, 3, 10).

• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer

… 200 ng of pertussis toxin (List Biological Laboratories, Inc., Campbell, CA, USA) was injected intraperitoneally (i.p.) on day 0 and 2 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)

… Non-induced age-matched littermate females were used as controls and were injected subcutaneously with an emulsion of PBS in CFA (Difco Laboratories, Detroit, USA), and with PTX (List Biological Laboratories, Inc., Campbell, CA, USA) at the same concentration and time points as the EAE animals …

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)