The virus control was prepared by adding 10 µL of EBOV/Mak in a tripartite soil load12 ,13 (link) (102 to 104 TCID50 virus; 0.25% bovine serum albumin [BSA, Sigma], 0.35% tryptone [Becton Dickinson], 0.08% bovine mucin [Sigma]) to 990 µL of VCM. Final concentrations in the control were: virus (102 to 104 TCID50/mL), BSA (0.0025%), tryptone (0.0035%), and mucin (0.0008%). The positive control were diluted in VCM using a ten-fold dilution scheme from 100 (undiluted) to 10−3, and 50 µL of the resulting solutions were added to Vero E6 cells. Cells were scored for CPE 14 days post-inoculation.
Bovine mucin
Manufactured by Merck Group
Sourced in France, Japan
Bovine mucin is a naturally derived, high-molecular-weight glycoprotein extracted from bovine sources. It is a key component of the mucus layer that lines various organs and tissues in the body. Bovine mucin exhibits unique physical and chemical properties that make it useful as a laboratory reagent for various applications.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Avantor (2 mentions)
Tryptone, by Avantor (2 mentions)
Mucin, by Merck Group (2 mentions)
K2hpo4, by Thermo Fisher Scientific (2 mentions)
Sodium chloride (nacl), by Avantor (2 mentions)
Nh4cl, by Avantor (2 mentions)
Mgcl2 7h2o, by Thermo Fisher Scientific (2 mentions)
Nh2 2co, by Avantor (2 mentions)
Cacl2, by Avantor (2 mentions)
Nahco3, by Thermo Fisher Scientific (2 mentions)
Potassium chloride (kcl), by Avantor (2 mentions)
Kh2po4, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Tryptone, by BD (1 mentions)
Tryptone, by Merck Group (1 mentions)
Mucin from bovine submaxillary glands, by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline dpbs 10, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane filter, by Merck Group (1 mentions)
0.22 μm pore, by Merck Group (1 mentions)
6 protocols using bovine mucin
1
Ebola Virus Control Preparation
2
Mucin-Based Soil Load Preparation
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A 3 mg/mL solution of mucin from bovine submaxillary glands (Merck Sigma-Aldrich, Darmstadt, Germany) was prepared in 1× phosphate-buffered saline (PBS) [74 ]. Soil load was also prepared following ASTM standards [61 ]; i.e., 0.5 g of tryptone (Merck Sigma-Aldrich), 0.5 g of bovine serum albumin (BSA, Merck Sigma-Aldrich), and 0.04 g of bovine mucin (Merck Sigma-Aldrich) in 10 mL of PBS, sterilized by passage through a 0.22 µm filter and stored at −20 °C.
3
Mammary Pheromone Analysis Protocol
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The mammary pheromone (MP), i.e., 2-methylbut-2-enal (2MB2; CAS # 497-03-0) and the 2-methylpent-2-enal (2MP2, CAS # 623-36-9), L-glutathione reduced (CAS # 70-18-8), mucin from bovine submaxillary glands (bovine mucin, CAS # 84195-52-8) and sterile filtered Dulbecco’s Phosphate Buffered Saline (DPBS 10×) were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France).
4
Standardized SARS-CoV-2 Inoculum Matrices
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Low and high inoculum levels were developed in three separate inoculum matrices at a target concentration of 3.5 and 7.0 log PFU on surfaces, respectively. Sterile 1× phosphate-buffered saline (PBS), pH 7.4, was prepared by adding 100 μL of diluted Φ6 stock (high, 10-fold dilution; low, 1,000-fold dilution) to 5 mL of PBS. Similarly, 100 μL of diluted Φ6 stock (high, 10-fold dilution; low, 1,000-fold dilution) was added to 5 mL of artificial saliva consisting of 1.54 mM KH2PO4 (Sigma-Aldrich), 2.46 mM K2HPO4 (Fisher Scientific, Loughborough, UK), 0.04 mg/L MgCl2·7H2O (Alfa Aesar, Ward Hill, MA), 0.11 g/L NH4Cl (VWR), 0.12 g/L (NH2)2CO (VWR), 0.13 g/L CaCl2 (VWR), 0.19 g/L KSCN (Acros Organics, Carlsbad, CA), 0.42 g/L NaHCO3 (Fisher Scientific), 0.88 g/L NaCl (VWR), 1.04 g/L KCl (VWR), and 3 g/L mucin (Sigma-Aldrich) at pH 7 (30 , 31 (link)). Lastly, the tripartite matrix (5 mL) was prepared as described in international standard ASTM E2197-17 (24 ) by combining 3.4 mL of PBS containing Φ6 stock (low and high) with a 1.6-mL solution consisting of 0.8 mg/mL bovine mucin (Sigma-Aldrich, St. Louis, MO), 2.5 mg/mL bovine serum albumin (VWR), and 3.5 mg/mL tryptone (VWR) to mimic fluids shed by infected individuals (1 (link), 3 (link), 24 , 32 (link)).
5
Artificial Saliva and Fecal Matrix Preparation
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The artificial saliva matrix was chosen to mimic cough or sneeze ejecta and prepared according to ASTM E2721-16. In brief, the artificial saliva matrix consisted of 0.21 g/L KH2PO4 (Sigma-Aldrich, St. Louis, MO), 0.43 g/L K2HPO4 (Fisher Scientific, Loughborough, UK), 0.04 mg/L MgCl2·7H2O (Alfa Aesar, Ward Hill, MA), 0.11 g/L NH4Cl (VWR), 0.12 g/L (NH2)2CO (VWR), 0.13 g/L CaCl2 (VWR), 0.19 g/L KSCN (Acros Organics, Carlsbad, CA), 0.42 g/L NaHCO3 (Fisher Scientific), 0.88 g/L NaCl (VWR), 1.04 g/L KCl (VWR), and 3 g/L mucin (Sigma-Aldrich) at pH 7 (ASTM International, 2016 ; Owen et al., 2021 (link)). The tripartite matrix was chosen to mimic fecal material shed by infected individuals and prepared as per international standard ASTM E2197-17 (ASTM International, 2017 ). In brief, the tripartite matrix consisted of 0.8 g/L bovine mucin (Sigma-Aldrich), 2.5 g/L bovine serum albumin (VWR), and 3.5 g/L tryptone (VWR) (ASTM International, 2017 ; Kasloff et al., 2021 (link); Riddell et al., 2020 (link); Sattar et al., 2003 (link)). Phi6 stock was added to each matrix to obtain inoculum levels of approximately 7 log PFU/mL.
6
Imaging Virus Movements on Mucin-Coated Surfaces
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For the imaging analyses of virus movements, the glass bottoms of culture dishes (Matsunami, Kishiwada, Japan) were coated with bovine mucin (1 mg/ml; Sigma-Aldrich, St. Louis, MO, USA), which was dissolved in phosphate-buffered saline (PBS) and filtered through a polyvinylidene fluoride membrane filter with a 0.22-μm pore (Millipore, Billerica, MA, USA) to remove insoluble mucin, and the dishes were then incubated for 1 h at room temperature. The glass surface of each dish was washed twice with PBS to remove any unbound mucin. This cycle of coating and washing was repeated twice. To examine virus movement, viruses diluted in PBS were added to the mucin-coated glass surfaces and observed by SRICM (Nikon, Tokyo, Japan) using a 100× lens objective (APO TIRF; numerical aperture, 1.49). Virus images were acquired every 1 s for a total duration of 15 min. Images of 50 filamentous AA, 30 spherical AA, and 30 spherical Taylor viruses were analyzed. Virions with lengths of more than 0.5 μm were regarded as filamentous, whereas virions with diameters of less than 0.3 μm were regarded as spherical.
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