Citations

Bioreagents:

… Polyclonal anti-botulinum neurotoxin type A [#730A, -BoNT-A pAb (chicken IgY)], monoclonal anti-botulinum neurotoxin type A [#731A, -BoNT-A mAb (mouse IgG)], botulinum neurotoxin type A toxoid [#133L, BoNT-A], and recombinant botulinum neurotoxin type B light chain [#620A, BoNT-B were obtained from List Biological Laboratories. All lyophilized bioreagents were reconstituted and stored per manufacturer recommendations.

• Product #730A – Anti-Botulinum Neurotoxin, Type A (Chicken lgY)
• Product #731L – Anti-Botulinum Neurotoxin, Type A (Mouse lgG monoclonal F1-40), Frozen Liquid
• Product #133L – Botulinum Neurotoxin Type A Toxoid from Clostridium botulinum
• Product #620A – Botulinum Neurotoxin Type B Light Chain, Recombinant

Botulinum toxins (BoNTs) and general reagents:

…  Serotypes A, B, and E were also purchased from List Biological Laboratories as complex toxins and BSA was added during the reconstitution step of the lyophilized powder supplied, as recommended by the manufacturer. …

Table1

Table showing primary and secondary antibodies:

… Goat anti-CtB, List Laboratories 703, …

Immunouorescencestaining:

Tissues were obtained from a previous study [1].  We evaluated with immunolabeling (Table1) whether neurotrophin-3 treatment regulates Sema4C expression specically in TrkC+ afferent neurons, which include proprioceptive afferents. …

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

Drugs and reagents:

… Pertussis toxin (PTX) was from List Biological Laboratories, Inc. (Campbell, CA, USA). …

Dynamic mass redistribution assay:

Antagonist properties of ligands were measured by assessing the concentration response curve to NPS in the absence and in presence of a fixed concentration of compound. FR900359 was added 60 minute before NPS, rolipram was incubated for 90 minute before NPS, while PTX was added 24 hours before NPS. Maximum picometer (pm) modifications (peak measured at 60 minute time point) were used to determine agonist response after baseline normalization.

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)

Materials:

Cholera toxin was purchased from List Biological Laboratory, Inc, California, USA; …

Induction of fluid accumulation by cholera toxin (CT):

Fluid accumulation in mouse intestinal loops in response to CT was performed in ARH1^+/+ and ARH1^-/- mice [28, 39]. After mice were anesthetized, intestine was exteriorized through a midline incision. Two or three intestinal segments of about 4 cm length each were generated by ligation with nylon suture, and 0.2 ml of PBS or PBS containing either 0.5 µg CT or 5 mM 8-Bromo-cAMP were injected into each loop [28]. …

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

Indirect immunofluorescence analysis (IFA):

…To resolve the C-terminal topology of TgATPaseP-GC, fresh extracellular parasites were stained with rabbit -HA (1:3000) and mouse -TgSag1 (1:10000) or rabbit -TgGap45 (1:8000) and -TgSag1 (1:10000) antibody combinations before and after permeabilization as described elsewhere (Blume et al. 2009). Briefly, 5 x 104 tachyzoites were released on BSA-coated coverslips and fixed with 4% PFA including 0.05% glutaraldehyde. Permeabilized coverslips were subjected to immunofluorescence assay as indicated above; however, all solutions were substituted to PBS for non-permeabilized staining as principle of the assay as shown in Figure 2B. To find the exact localization of TgATPaseP-GC protein, the IMC was separated from the PM by treating extracellular parasites with -toxin from Clostridium septicum (20 nM, 2 h) (List Biological Laboratories, US) followed by fixation on BSAcoated coverslips. In both cases, the standard secondary antibody staining procedure was performed afterwards, as described for immunofluorescence assay …

Human blood sample processing and in vitro stimulation:

… Human blood was anti-coagulated with 20 units/ml pyrogen-free sodium heparin (American Pharmaceutical Partners, Inc.; Schaumberg, IL). All blood products were kept at room temperature and processed within 4h from collection. For human whole blood assays, neonatal cord blood or adult whole blood (WB) was first diluted with sterile RPMI 1640 medium (Invitrogen; Carlsbad, CA) and 175l of the diluted WB was added to each well of a 96 well U-bottom plate (Becton Dickinson; Franklin Lakes, NJ, USA) containing 75l of RPMI, freshly prepared FIM proteins, or licensed vaccines at 10 the final concentration. The same formulations were used for both in vivo and in vitro assessment, so the FIM2/3 formulation was prepared by mixing dilutions of purified FIM2/3 (200g/ml, List Biological Laboratories, Inc.) in PBS, which at 1/10 human dose would correlate with 5g/250l. …’

• Product #186 – Fimbriae 2/3 from B. pertussis

Intestinal organoid culture and live imaging:

Primary intestinal epithelial organoids were grown as described before⁵⁶. Briefly, the small intestine and colon were flushed and cut into small pieces that were dissociated in PBS containing 2mM EDTA for 30min at 4C. After extensive washing, the isolated crypts were pelleted and mixed with 25l of Matrigel (Corning) and put in a 24-well plate. After polymerization of the Matrigel, complete culture medium containing advanced DMEM/F12 (Gibco) supplemented with B27 supplement (0.02%, Invitrogen), N2 supplement (0.1%, Invitrogen), N-acetylcysteine (0.0025%, Sigma-Aldrich), mouse epidermal growth factor (mEGF; 0.001%, Invitrogen), and conditioned Rspondin and mNoggin medium was added to the wells. Organoids were seeded and imaged in an 8-well chamber (iBidi) for cell death analysis by real time lapse microscopy or in 24-well plates for Western blotting analysis. Cell death was induced with TcdA (1g/ml, Enzo Life Sciences), TcdB (1g/ml, List Laboratories), …

• Product #155 – Toxin B from Clostridium difficile

Toxin preparation:

C. difficile toxin was prepared as previously described (87). Briefly, C. difficile strain r20291 (AB and AB⁺) was cultured in tryptone-yeast extract media for 24 hours in an anaerobic chamber at 37C. Cultures were diluted to an OD that corresponds to 2 10⁷ CFU/ml and spun down; the supernatant was sterilized using 0.22 M filters. Supernatant prepared from toxin-negative (control) and toxin-positive strain was used to stimulate BMDMs at a 1:5 dilution.

Purified TcdB was obtained from List Biological Laboratories (catalog 155) and used at final concentration of 0.5 g/ml, as previously described (7).

• Product #155 – Toxin B from Clostridium difficile

… Bordetella pertussis Adenylate Cyclase Toxin/toxoid (Act) was purchased from Sigma (Cat#A0847, Sigma, St. Louis, MO), and List Biological Laboratories (Cat#188 & 189, List Biological Laboratories Inc. …

• Product #188L – Adenylate Cyclase Toxin, Recombinant from B. pertussis