RV strains used were simian RV SA11 strain (G3P[2]) and bovine RV RF strain (G6P[1]) and were rescued using reverse genetics (detailed below) (42 (link), 71 (link), 95 (link), 96 (link)). The natural isolate of the RF strain was a kind gift from the laboratory of Dr. Ulrich Desselberger (University of Edinburgh, UK). Viruses were propagated in MA104 cells cultured in DMEM supplemented with 0.5 μg/mL porcine pancreatic trypsin type IX (Sigma-Aldrich), and viral titers were measured by plaque assay as described below.
Porcine pancreatic trypsin type 9
Manufactured by Merck Group
Sourced in United States
Porcine pancreatic trypsin type IX is a laboratory enzyme derived from the pancreas of pigs. It is a proteolytic enzyme that catalyzes the hydrolysis of peptide bonds in proteins, particularly those involving the carboxyl group of arginine or lysine residues.
Automatically generated - may contain errors
Lab products found in correlation
Medium 199, by Thermo Fisher Scientific (2 mentions)
Ma104 clone 1 cells, by American Type Culture Collection (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Zellshield, by Minerva Biolabs (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Ab35958, by Abcam (1 mentions)
Amphotericin, by Thermo Fisher Scientific (1 mentions)
Heparin sepharose, by GE Healthcare (1 mentions)
Sepharose 4b, by GE Healthcare (1 mentions)
Fsllry nh2, by Bio-Techne (1 mentions)
Cyanogen bromide activated, by GE Healthcare (1 mentions)
Tfllrn nh2, by Bio-Techne (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
6 protocols using porcine pancreatic trypsin type 9
1
Reverse Genetics for Rotavirus Strains
2
Propagation and Titration of Human Rotaviruses
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African green monkey kidney epithelial cells (MA104) and human epithelial colorectal adenocarcinoma cells (Caco-2) were propagated in Dulbecco's Modified Eagle Medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 1% (v/v) Zell Shield (Minerva Biolabs, Berlin, Germany) and heat inactivated, 10% (v/v) fetal bovine serum (Sigma). Human rotavirus (HRV) strains Wa (ATCC® VR-2018), WI61 (ATCC® VR-2551), HRV 408 (ATCC® VR-2273), HRV 248 (ATCC® VR-2274), DS-1 (ATCC® VR-2550) were purchased from ATCC (American Type Culture Collection, Rockville, MD, USA); virus was activated with 5 µg/ml of porcine pancreatic trypsin type IX (Sigma) for 30 min at 37 °C and propagated in MA104 cells using DMEM containing 0.5 µg of trypsin per ml as described previously [54] (link). Viral titers are expressed as focus-forming unit (FFU) per ml.
3
Propagation and Quantification of Clinically Relevant Viruses
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The neurovirulent strains LV (21 (link)) and MS (ATCC VR-540) of HSV-1 and HSV-2, were propagated in Vero cells at 37°C (22 (link)). HRhV 1A (ATCC VR-1559) was propagated in HeLa cells, at 33°C. HSV-1, HSV-2, and HRhV titers were determined by the standard plaque method and expressed as plaque-forming unit (PFU)/ml. A bacterial artificial chromosome (BAC)-derived HCMV strain Towne incorporating the green fluorescent protein (GFP) sequence (23 (link)) was propagated on HFF-1 and viral titres were determined by fluorescent focus assay. RSV strain A2 (ATCC VR-1540) was propagated in Hep-2 and titrated by the indirect immunoperoxidase staining procedure using an RSV monoclonal antibody (Ab35958, Abcam) (24 (link)). Human HRoV strain Wa (ATCC VR-2018) was activated with 5 μg/ml of porcine pancreatic trypsin type IX (Sigma) for 30 min at 37°C and propagated in MA104 cells by using MEM containing 0.5 μg of trypsin per ml. HCMV, RSV and HRoV titers were expressed as focus-forming units (FFU)/ml. Virus stocks were maintained frozen (-80°C).
4
Simian Rotavirus SA11 Infection Model
5
Isolation and Characterization of PAR2
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Frozen or fresh human adult lung and skin samples, ranging from 15 to 20 g, were purchased through the Collaborative Human Tissue Network; cyanogen bromide–activated Sepharose 4B from GE Healthcare; heparin-Sepharose, Na-benzoyl-dl -arginine 4-nitro-anilide, porcine pancreatic type IX trypsin (13,000–20,000 U/mg), sulphinpyrazone, calcium ionophore (A23187), porcine intestinal mucosa heparin (mean mol wt 16 kD), SBTI, cellulose phosphate (fibrous form), and leupeptin from Sigma-Aldrich; FCS, DMEM, penicillin, streptomycin, and amphotericin from Thermo Fisher Scientific; anti-PAR2 IgG and anti-EMR2 from R&D Systems; and PAR2-activating peptide (SLIGRL-NH2), PAR1-activating peptide (TFLLRN-NH2), PAR2 peptide control LRGILS-NH2, and PAR2 antagonist (FSLLRY-NH2) from Tocris Bioscience. PAR2 agonists or antagonist were diluted in a buffer comprising 20% DMSO and 80% PBS.
6
Simian Rotavirus SA11 Infection Model
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Simian RV strain SA11 (4F derivative, provided by Dr. John T. Patton, National Institutes of Health) was used to infect monkey kidney MA104 clone 1 cells (American Type Culture Collection) as described by Arnold et al., 2009 (Arnold et al., 2009 ). Cells were sub-cultured at 37°C and 5% CO2 in Medium 199 (Life Technologies) supplemented to contain 0.25 µg/ml Amphotericin B, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum. SA11 was activated prior to infection by incubation at 37°C for 1 hour in 10 µg/ml of porcine pancreatic type IX trypsin (Sigma Aldrich).
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