Bacterial Toxin Research Citations

Cell isolation and propagation

The uterine horns were collected from one 8-week-old C57BL female mice (Wuhan University Center for Animal Experiment). The Animal Ethics Committee of Wuhan University Center for Animal Experiment approved this study. Cell isolation and culture procedures were carried out according to the previous studies with minor modifications [24, 25, 29, 30]. In brief, fresh murine uterus from one female mouse was dissected and the uterine horns were collected as illustrated in Fig. 1A. Uterine horns were minced into pieces and dispersed into single cells by digestion with collagenase (StemCell, Vancouver, BC, Canada) plus trypsin. The primary mouse endometrial epithelial cells were co-cultured with irradiated mouse fibroblast 3T3 cells (J2 strain) (YongTech, Shenzhen, China) in primary epithelial culture basic medium (PECBM). PECBM contains DMEM and nutrient F-12 Ham (3:1) (v/v) (Sigma-Aldrich), supplemented with 5% FBS (GIBCO), 2 nM triiodothyronine (Sigma), 0.5% insulin–transferrin–selenium reagent (Life Technologies), 5 μg/ml transferrin (Life Technologies), 10 ng/mL epidermal growth factor (Sigma), 0.4 μg/mL hydrocortisone (Sigma), 1 nM cholera toxin (List Biological Labs), 0.5 μg/mL amphotericin B (Fungizone; Bristol-Myers Squibb), 40 μg/mL gentamicin (Gentacin; Life Technologies), and 5 to 10 mol/L Y-27632 (Enzo Life Sciences). …

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

… cholera toxin (List Biological Labs, Campbell, CA) in a humid environment with 5% CO2 at 37°C. …

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

… On days 0 and 2, the immunized mice received additional ip injections of 200 ng pertussis toxin (List Biological Labs). Starting 1 wk after the first immunization, EAE …

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)

2.6 Immunocytochemistry

The following process was based on previously published procedures in the lab (Aicher et al., 2013; Hegarty et al., 2018). Prior to immunocytochemical processing, fixed stellate ganglia (SG) were rinsed with 0.1 M PB followed by 0.1 M Tris-buffered saline pH 7.6 (TS) and placed in 0.5% bovine serum albumin (BSA, Sigma-Aldrich) in TS for 30 min. SG were then incubated for 48 h at 4°C in primary antibody solution of goat anti-cholera toxin b (CTb) (1:25000, cat. 703, List Biological, RRID: AB_10013220) (Experiment 1), or goat anti-vesicular acetylcholine transporter (VAChT) (1:1000, cat. ABN100, EMD Millipore, RRID: AB_2630394) (Experiment 2), in 0.25% Triton X-100 (Sigma)/0.1% BSA in 0.1 M TS. SG were then rinsed and incubated for 2 h at room temperature in donkey anti-goat Alexa Fluor (AF) 647 secondary antibody (1:800, cat. 705–605–147, Jackson ImmunoResearch, RRID: AB_2340437) in 0.1% BSA in TS. SG were rinsed, whole mounted, and cover-slipped using Prolong Gold Antifade mountant (ThermoFisher). The goat anti-CTb antibody has been used extensively to detect the beta subunit of cholera toxin (Hegarty et al., 2010, 2018) and preadsorption of this antibody with CTb abolished immunolabeling in rat and rabbit spinal cord sections (Llewellyn-Smith et al., 1995). The VAchT antibody has been used extensively in recent rodent studies to identify cholinergic neurons using immunocytochemistry (Avila et al., 2020; Webber et al.,) and VAchT protein levels using Western blotting (Li et al., 2021; Yokoi et al., 2021).

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

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