Three milliliters of sterile molten 0.5% (w/v) agarose containing 0.5% (w/v) of mucin (porcine type III, Sigma M1778) was overlaid onto sterile microscope slides (76 × 26 mm) rapidly. The sterile slide was placed into a petri dish and the agarose left to solidify, after which 3-mm-diameter holes were punched out of the medium. Ten microliters of spores (1 × 1010/mL) was added to the wells. The petri dished was closed and sealed with parafilm and incubated for 48 hours at 37°C. Before staining with amido black solution for 30 minutes (Sigma A8181), the agarose was compressed to a thin layer using a 500-g weight (30 minutes with Whatman No. 3 between agarose and weight). The slide was then de-stained several times with 7% (v/v) acetic acid.
M1778
Manufactured by Merck Group
Sourced in United States
M1778 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of M1778 is to provide a reliable and consistent tool for various scientific applications. Further details on the intended use or specific features of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Epoch 2 plate reader, by Agilent Technologies (2 mentions)
Amido black solution, by Merck Group (1 mentions)
Whatman no 3, by Merck Group (1 mentions)
Balb c mice, by Jackson ImmunoResearch (1 mentions)
Omni th, by Omni International (1 mentions)
Starch, by Merck Group (1 mentions)
Tamarind xyloglucan, by Megazyme (1 mentions)
Cellometer cell counter, by Revvity (1 mentions)
Glacial acetic acid, by Avantor (1 mentions)
Glucose, by Merck Group (1 mentions)
P0447, by Agilent Technologies (1 mentions)
Potassium dihydrogen orthophosphate, by Thermo Fisher Scientific (1 mentions)
Pvp360, by Merck Group (1 mentions)
Terrific broth, by Merck Group (1 mentions)
12 protocols using m1778
1
Spore Adhesion Assay with Mucin-Agarose
2
Cultivation of Akkermansia muciniphila
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Akkermansia muciniphila MucT (=DSM 22959 T) was obtained from the German Collection of Microorganisms and Cell Cultures (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). A. muciniphila was cultivated anaerobically at 37 °C for 48 h in two different types of media: brain–heart infusion medium (BHI; 237500, BD, Hampton, NJ, USA) and a basal medium. The basal medium contained (L−1); 16 g soypeptone, 25 mM glucose, 0.4 g KH2PO4, 0.53 g Na2HPO4, 0.3 g NH4Cl, 0.3 g NaCl, 0.1 g MgCl2.6H2O, 0.11 g CaCl2, 4 g NaHCO3, 0.5 g cysteine, 1 mL alkaline trace element solution, 1 mL acid trace element solution, and 1 mL vitamin solution. The alkaline trace element, acid trace element, and vitamin solutions were prepared as described previously9 (link). The media were supplemented with 0.2% (w/v) mucin from porcine stomach (M1778, Sigma-Aldrich, St. Louis, MO, USA), purified via ethanol precipitation55 (link). All procedures for media preparation were performed under anaerobic conditions in serum bottles sealed with butyl-rubber and gas phase N2.
3
Evaluation of Colistin and PPMO Combination in Murine E. coli Sepsis
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Briefly, 7- to 8-week-old female BALB/c mice (Jackson Laboratory, Sacramento, CA, USA) were infected intraperitoneally (i.p.) with 8 × 104 CFU of E. coli (MRSN 388634) in DPBS with 5% mucin (M1778; Sigma-Aldrich). At 2 and 6 h postinfection, 0.25 mg/kg colistin sulfate, 100 µg PPMO, or the combination was administered i.p. in 100 µl DPBS. Morbidity was assessed at 24 h, immediately prior to euthanasia. Mice were scored from 0 to 6 as follows: 0 for the absence or 1 for the presence of a ruffled coat; 0 for a normal posture, 1 for a hunched posture, and 2 for a prostrate posture; and 0 for nondisturbed movement, 1 for slow movement, 2 for movement after prodding, and 3 for no movement. Mice that succumbed to infection prior to euthanasia were scored as the maxima, 6 (moribund). Spleen and liver were harvested and homogenized in 1 ml DPBS (Omni TH; Omni International, Kennesaw, GA), serially diluted, and plated on blood agar.
4
Artificial Saliva Composition Protocol
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Artificial saliva (AS) was prepared based on a Phosphate-buffered saline (PBS) in which porcine gastric mucin (M1778, Sigma-Aldrich, Saint Louis, MO, USA) at a concentration of 10 g/L and xanthan gum (G1253, Sigma-Aldrich, Saint Louis, MO, USA) at a concentration of 4 g/L were dissolved.
5
Mucin-Supplemented Pneumococcal Infection
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For each strain of S. pneumoniae (E611, E674, E676, E678, E683, E684, and ATCC 49619), two groups of neutropenic mice received the bacterial inoculum without or with mucin supplementation to test its impact on the lethality and reproducibility of the model. When supplemented, the bacterial inoculum was mixed with porcine mucin (M-1778; Sigma Chemical Company, St Louis, MO) just before nasal instillation. For this instance, a mucin stock solution (10% [wt/vol]) was diluted 1:1 with the pneumococcal suspension in supplemented THB with ~8 log10 CFU/mL prepared as describe above, for a final mucin concentration of 5%.
6
Artificial Saliva Preparation Protocol
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7
Culturing Human and Mouse Bacteroides
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Bacteroides ovatus (MDA-HVS BO001) was isolated and cultured from healthy volunteer’s stool samples in a Whitley anaerobic chamber (10% H2, 5% CO2 and 85% N2). Human-derived B. ovatus (ATCC 8483) and human-derived B. theta (ATCC 29148) were purchased from American Type Culture Collection (ATCC). Mouse-derived BT (MDA-JAX BT001) was previously isolated15 (link). Bacterial number was quantified using a Nexcelom Cellometer cell counter with SYTO BC dye and propidium iodide. Bacterial growth experiments were performed in a liquid media, BYEM10, composed of a hybrid of BHI and M10 supplemented with yeast extract as previously described15 (link),45 (link). Bacteria were cultured up to 24 or 48 hours at a starting concentration of 1 × 106 bacteria/ml in BYEM10 broth (pH 7.2) with or without 5 mg/ml of porcine gastric mucin (M1778, Sigma-Aldrich), wheat arabionoxylan (wheat flour; low viscosity; Megazyme), xylan (Beechwood; Megazyme), xyloglucan (Tamarind; Megazyme), or starch (wheat; Sigma-Aldrich). Optical densities (OD600 nm) of bacterial cultures were measured with a BioTek Epoch 2 plate reader.
8
Mucin Degradation Assay for Pathogenicity
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To ascertain mucin degradation capacity, 1.5% (w/v) agar plates with 0.3% (w/v) mucin from porcine stomach M1778 (Sigma-Aldrich, St. Louis, Missouri, USA) and glucose 1% (w/v) (Sigma-Aldrich) were used [13 (link)]. Following incubation at 37 °C for 72 h, the mucin plates were flooded with 2 mL of 0.1% amido black 10B (Alfa Aesar, Haverhill, MA, USA), and prepared with 3.5 M glacial acetic acid (VWR Chemicals). After 30 min, mucin plates were washed twice with 1.2 M acetic acid, and halo formation around colonies (as indicator of pathogenicity) was recorded.
9
Quantifying Protein-Ligand Interactions via ELISA
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High binding enzyme-linked immunosorbent assay (ELISA) plates were coated with ligands conjugated to human serum albumin (Dextra, Reading, United Kingdom) at a concentration of 10 µg/mL overnight at 4°C. For mucin, porcine stomach type III (M1778, Sigma), a solution was made fresh in PBS (pH 7.4) and used to coat plates at 1 µg/mL (overnight at 4°C). After blocking the plate with 2% (v/v) bovine serum albumin (BSA) in PBS (1 hour at 30°C), recombinant proteins (rCotEN or rCotEC) were diluted in 1% (v/v) BSA at a concentration of 0.5 μM and incubated for 2.5 hours at 30°C. rCotEN (mouse) and rCotEC (mouse) were used as primary antibodies and anti-mouse immunoglobulin G–horseradish peroxidase (DAKO P0447) was used as the secondary antibody.
10
Isolation and Cultivation of Gut Microbiome
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