Madin–Darby canine kidney (MDCK) and swine testicle (ST) cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (Invitrogen, Waltham, Massachusetts, USA). DMEM infecting media containing 0.3% bovine serum albumins (BSA) (Sigma-Aldrich, St. Louis, Missouri, USA), 1% antibiotic-antimycotic, and 0.5 or 1 μg/mL of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich) were used for virus infection of cells. Influenza B/Brisbane/60/2008 (Victoria lineage) was provided by the Centers for Disease Control and Prevention (CDC). Influenza D/bovine/Kansas/1-35/2010 was isolated from bovine nasal swab samples [30 (link)]. IBV was amplified in MDCK cells using DMEM infecting media containing 1 μg/mL TPCK-treated trypsin at 33 °C with 5% CO2 for 4 days. IDV was propagated in ST cells using DMEM infecting media containing 0.5 μg/mL TPCK-treated trypsin at 37 °C with 5% CO2 for 4 days. Virus loads in nasal swabs and bronchoalveolar lavage fluid (BALF) samples were determined on MDCK cells by indirect immunofluorescence assay (IFA). A 50% tissue culture infective dose (TCID50) was calculated to determine virus titers using Reed and Muench method [40 (link)].
N tosyl l phenylalanine chloromethyl ketone tpck treated trypsin
Manufactured by Merck Group
Sourced in United States
N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin is a proteolytic enzyme used in laboratory settings. It is derived from bovine pancreas and has been chemically modified with TPCK to enhance its specificity and activity. The core function of TPCK-treated trypsin is to cleave peptide bonds at the carboxyl side of arginine and lysine residues in proteins.
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Lab products found in correlation
Trypsin, by Merck Group (3 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Bovine serum albumins, by Merck Group (1 mentions)
Antibiotic antimycotic, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Lt1 transfection reagent, by Mirus Bio (1 mentions)
Eclipse ti s, by Nikon (1 mentions)
Nis element, by Nikon (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by MP Biomedicals (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Antibiotic antimycotic solution, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
16 protocols using n tosyl l phenylalanine chloromethyl ketone tpck treated trypsin
1
Influenza B and D Virus Propagation
2
Virus Microneutralization Assay Protocol
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Virus microneutralization assays (MNAs) were performed as previously described [19 (link)]. Briefly, KPF1-HEK or KPF1-Antx hMAbs, or IgG1 isotype controls, were 2-fold serially diluted in PBS in 96 well plates (starting concentrations of 200 µg/mL). One hundred plaque forming units (PFUs) of each virus (Brisbane/H1N1, pH1N1, NC/H1N1, WI/H1N1, GA/H1N1, and Brisbane/H3N2) were then added to the hMAb dilutions and incubated for 1 h at room temperature. MDCK cells (96 well plate format, 5 × 104 cells/well, quadruplicates) were then infected with the hMAb-virus mixture for 1 h at room temperature. After viral adsorption, cells were maintained in p.i. medium, with 1 µg/mL of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich, St. Louis, Missouri, MO, USA), and incubated at 37 °C. Virus neutralization was determined by crystal violet staining at 72 h p.i. The neutralization titer 50 (NT50) was determined by a sigmoidal dose response curve (GraphPad Prism, v7.0) [19 (link)]. Mock-infected cells and viruses in the absence of the KPF1 hMAbs were used as an internal control.
3
Viral Titer Determination in MDCK Cells
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MDCK cells were inoculated with 10-fold serial dilutions of virus stocks, nose swabs, throat swabs, or homogenized tissue samples. The cells were washed with phosphate-buffered saline (PBS) 1 h after inoculation and cultured in infection medium, consisting of EMEM supplemented with 100 U/ml PEN, 100 U/ml STR, 2 mM l -Glu, 1.5 mg/ml NaHCO3, 10 mM HEPES, 1× NEAA, and 20 μg/ml trypsin (N-tosyl-l -phenylalanine chloromethyl ketone [TPCK]-treated trypsin; Sigma). Three days after inoculation, supernatants of cell cultures were tested for agglutinating activity using TRBCs as an indicator of virus replication. Infectious virus titers were calculated from 4 replicates each of the homogenized tissue samples, nose swabs, and throat swabs and from 10 replicates of the virus stocks by the method of Reed and Muench (45 ).
4
Reverse-engineered 1918 Influenza Virus
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Influenza virus A/South Carolina/1918 (H1N1) was generated by reverse genetics (9 (link)) and handled in biosafety level 4 (BSL-4) containment at the National Microbiology Laboratory (NML). Sequences of the 1918 influenza viral segments were based on data reported under GenBank accession numbers DQ208309 , DQ208310 , DQ208311 , AF117241 , AY744935 , AF250356 , AY130766 , and AF333238 . 1918 influenza virus was cultured using Madin-Darby canine kidney (MDCK; ATCC, Manassas, VA, USA) cells. MDCK cells were grown in minimum essential medium (MEM; HyClone) supplemented with 5% fetal bovine serum (FBS; HyClone) and 1× l -glutamine (l -Glu; Gibco, Life Technologies, Grand Island, NY, USA).
A passage 2 (P2) virus stock was prepared using MEM supplemented with 0.1% bovine serum albumin (BSA) (fraction V; HyClone), 1×l -glutamine, and 1 μg/mL N-tosyl-l -phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich). This stock was used for animal inoculation. The mouse 50% lethal dose (MLD50) for this stock was determined previously to be 103.2 PFU (9 (link)); this value was confirmed prior to the use of the stock for macaque infection.
A passage 2 (P2) virus stock was prepared using MEM supplemented with 0.1% bovine serum albumin (BSA) (fraction V; HyClone), 1×
5
Trypsin-mediated Protein Conformational Changes
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Limited proteolysis by trypsin was performed at 37°C in 20 mM HEPES, 150 mM NaCl, 2% DMSO (pH 8.2), with 0.15 μg/μL HMBS in the absence (DMSO control) or presence of 84 μM compound and 2% DMSO. The proteolysis was initiated by adding 1 μg/mL N-tosyl-l -phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich). After 30 min, aliquots were removed and transferred to Laemmli loading buffer containing 2 μg/mL soybean trypsin inhibitor. Samples resolved by SDS-PAGE with 10% Mini-Protean TGX gels (Bio-Rad Laboratories, Hercules, California) were analyzed using the Image Lab software (Bio-Rad). The unpaired t test (two-tailed) was performed using GraphPad Prism.
6
Rescue and Propagation of Recombinant Influenza Viruses
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To rescue the recombinant viruses 1 µg of each helper plasmid along with 100 ng of each RNA pol I plasmid for each gene were transfected with 10 µL LT1 transfection reagent (Mirus Bio, Madison WI, USA) in 200 µL OPTI-mem (HyClone) in 293GT cells. After 48 h, the supernatant was collected and treated with 1 µg/mL of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) treated trypsin (Sigma Aldrich, ON, Canada) for 30 min at 37 °C, blind passaged onto MDCK cells, and harvested after the appearance of cytopathic effect (CPE) 48–72 h later. Two versions of A/Anhui/1/2013 (H7N9) were rescued by reverse genetics, the wild-type version (H7N9-RG) and a version containing the PBC HA (H7N9-PBC). To generate viral stocks, MDCK cells in T150 cm2 flasks were washed with phosphate buffered saline (PBS), and infected with a low multiplicity of infection (MOI) of virus diluted in MEM supplemented with 0.1% bovine serum albumin (BSA, Gibco), L-Glu, 1 µg/mL TPCK treated trypsin (Sigma Aldrich, ON, Canada) for 48–72 h. Viruses were harvested when 80%–90% CPE was observed, and stocks were titered by plaque assays.
7
Plaque Assay for Low Pathogenic Avian Influenza Viruses
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Indicated viruses/samples were serially 10-fold diluted and MDCKII cells were incubated with virus dilutions at 37 °C/5% CO2. After one hour, the cells were washed twice with phosphate buffered saline (PBS) and covered by semi-solid agar in DMEM containing 4% bovine serum albumin (BSA). LPAIVs were incubated in the presence of 2 μg/mL of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma Aldrich). Plates were incubated at 37 °C for 3 days and fixed using 10% formaldehyde containing 0.1% crystal violet. Viral titres were expressed as plaque forming units per mL (PFU/ml). Moreover, plaque sizes were measured by microscopy (Eclipse Ti-S with the software NIS-Elements, version 4.0; Nikon). The mean plaque size for each recombinant virus (about 50 plaques each) was expressed as the percentage of the mean plaque size of the respective parental reverse genetic wild-type virus in the presence of trypsin.
8
Influenza A Virus Propagation and Titration
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Influenza A Virus A/Bayern/74/2009 (H1N1pdm09) (By09) was propagated on Madin-Darby canine kidney cells (MDCK II) cells in MEM containing 0.2% bovine serum albumin, 1 Unit/ml Penicillin, 1 µg/ml Streptomycin and 2 µg/ml N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich). For TCID50 assay, serial tenfold dilutions in infection medium were prepared and added to MDCK II cells on 96-well tissue culture plates. After incubation for three days at 37 °C and 5% CO2 each well was monitored for cytopathic effects. Viral titers were calculated according to Spearman-Kärber34 .
9
Generation of Recombinant Influenza Viruses
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Virus rescues were performed as we described previously [2 (link),40 (link),52 (link)]. Briefly, co-cultures (1:1) of 293T and MDCK cells in 6-well plates were co-transfected with 1 μg of each of the ambisense plasmids (pDZ-PB2, −PB1, −PAWT+ or PAMT−, −HA, −NP, −NA, −M, and NSWT+ or NS1MT−) using Lipofectamine 2000 (LPF2000, Invitrogen). At 12 h post-transfection (p.t.), the transfection medium was replaced with DMEM containing 0.3% bovine serum albumin (BSA), 1% P-S-G, and 0.5 μg/mL of N-tosyl-l-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma). At 3–4 days p.t., tissue culture supernatants (TCS) were collected, clarified, and used to infect fresh MDCK cells. At 3 to 4 days post-infection (p.i.), recombinant viruses were plaque purified and scaled up in MDCK cells. Stocks were titrated by immunofocus assay (Fluorescence Focus Units (FFU)/mL)) on MDCK cells [2 (link),52 (link)]. Virus stocks were confirmed by sequencing the PA and NS1 ORFs using purified total RNA (TRIzol reagent; Invitrogen) from infected MDCK cells according to the manufacturer’s specifications.
10
Influenza A Virus Propagation and TCID50 Determination
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Influenza A Virus A/Bayern/74/2009 (H1N1pdm09) (IAV) was kindly provided from the Federal Research Institute for animal health (FLI, Riems, Germany). MDCK II-cells were prepared in MEM-medium containing 14% bovine serum albumin (35%, MP Biomedicals, Eschwege Germany), 10000 Unit/ml penicillin/streptomycin (v/v, Gibco, Thermofischer, Darmstadt, Germany) and 2 µg/ml N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich/Merck, Darmstadt, Germany) for the cultivation of IAV. The calculation of TCID50/ml was performed by following the protocol of “Virology Methods Manual, p. 374”54 .
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