All chemicals, including tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin, were obtained from Thermo Scientific, unless otherwise stated. Acetonitrile (CH3CN, Honeywell Riedel-de Haen), formic acid (FA) and LC-MS grade water (Honeywell Riedel-de Haen) were obtained from Fisher Scientific Italia (Rodano, Milano). The tandem mass tag (TMT) TMT-sixplex isobaric mass tagging kit was purchased from Thermo Fisher Scientific (Rockford, IL, USA). The strain of P. digitatum was isolated in our laboratory and typified by the Spanish Type Culture Collection (CECT), Valencia, Spain. TBZ was obtained from Sigma Aldrich (St Louis, MO, USA). α-sarcin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). BE27 was purified as described [13 (link),39 (link),40 (link)].
Tpck treated trypsin
Manufactured by Thermo Fisher Scientific
Sourced in United States
TPCK-treated trypsin is a proteolytic enzyme used in cell culture and molecular biology applications. It is used to dissociate adherent cells from culture surfaces and separate cells within tissue samples. TPCK-treated trypsin has been modified to reduce chymotrypsin-like activity.
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Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
α sarcin, by Santa Cruz Biotechnology (1 mentions)
Acetonitrile ch3cn, by Honeywell (1 mentions)
Ldh assay, by Thermo Fisher Scientific (1 mentions)
Anti β actin antibody a5316, by Merck Group (1 mentions)
Gtx632604, by GeneTex (1 mentions)
Bright glo, by Promega (1 mentions)
Polyethylenimine (pei), by Merck Group (1 mentions)
Sybr green, by Bio-Rad (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Aprotinin, by Carl Roth (1 mentions)
22 protocols using tpck treated trypsin
1
Proteomic analysis of Penicillium digitatum
2
Influenza A Virus Propagation and Infection
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Influenza A virus (H1N1) strain A/PR/8/34 (PR8) and PR8-GFP were propagated in the allantoic cavity of 10-day-old embryonated specific-pathogen-free chicken eggs, and viral titers were determined by standard plaque assay on confluent monolayers of MDCK cells as previously described [62 (link)]. A549 cells were infected with PR8 virus at the given MOI and media only, used as mock control. After one hour of adsorption, the viral inoculum was discarded and replaced by Opti-MEM added with 0.5–1 μg/ml TPCK-treated trypsin (Thermo Fisher Scientific) in a 37°C incubator. Cells were harvested at select time points for different assays. Viral titers were determined by standard plaque assay on confluent monolayers of MDCK cells.
For mice infection, six-week-old female wild type, or Yap+/- C57BL/6J mice were anesthetized with pentobarbital prior to intranasal infection with 103 PFU of PR8 virus in 50 ml of PBS (Gibco) or mock infected. Lungs were harvested at indicated timepoints and homogenized for analysis. Mice were monitored daily for weight loss over a 5-day period.
For mice infection, six-week-old female wild type, or Yap+/- C57BL/6J mice were anesthetized with pentobarbital prior to intranasal infection with 103 PFU of PR8 virus in 50 ml of PBS (Gibco) or mock infected. Lungs were harvested at indicated timepoints and homogenized for analysis. Mice were monitored daily for weight loss over a 5-day period.
3
Influenza A Virus Infection of Macrophages and Fibroblasts
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After macrophage differentiation, cells were infected with the virus strain PR8 (influenza A virus/IAV/H1N1/Puerto Rico/8/1934) and a multiplicity of infection (MOI) 5. After washing the cells with phosphate-buffered saline (PBS, Thermo Fisher Scientific), they were incubated with the virus solution diluted in PBS (containing 0.2 % bovine serum albumin (BSA, Roth, Karlsruhe, Germany), 1 mM MgCl2 (Sigma Aldrich), and 0.9 mM CaCl2 (Sigma Aldrich)). After 30 min, the cells were washed with PBS and cultivated in Roswell Park Memorial Institute (RPMI, Thermo Fisher Scientific) medium (containing 0.2 % human serum albumin (HSA, PAN Biotech), 1 mM MgCl2, 0.9 mM CaCl2, and 30 ng/ml TPCK-treated trypsin (Thermo Fisher Scientific)) for 8 h and 24 h. Supernatants of infected hMDM were collected and used for standard plaque assay to determine the viral load and to determine cytotoxicity by using LDH assay (Thermo Fisher Scientific) according to the manufacturer’s protocol. PR8 was used to infect IMR-90s with an MOI 1; the cells were washed with PBS and incubated in virus solution diluted with PBS (containing 0.2 % BSA, 1 mM MgCl2, 0.9 mM CaCl2). After washing, the cells were cultivated in DMEM containing 0.2 % BSA, 1 mM MgCl2, 0.9 mM CaCl2, and 30 ng/ml TPCK-treated trypsin for 8 h and 24 h.
4
Virus Isolation and Titration
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To remove any bacterial contaminants before use, supernatants from nasal, oral, and rectal swabs were thawed, vortexed, and filtered through polyvinylidene difluoride membrane, which has low protein-binding ability (pore size, 0.45 μm; catalog no. SLHVJ13SL, Millipore, Billerica, MA, USA). Each sample was diluted ten-fold serially into serum-free MEM supplemented with 1 μg/ml TPCK-treated trypsin (Thermo Fisher Scientific) and antibiotics; 25 μl of each dilution was inoculated onto PBS-washed confluent MDCK cells in 96-well plates. After 5 days of incubation at 37 °C under 5% CO2, the TCID50 of each sample was calculated according to hemagglutination activity by using 0.55% red blood cells from guinea pigs.
5
SARS-CoV-2 Spike Protein Detection
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Chemicals were purchased from the following manufacturers: Bright-Glo, FuGENE HD transfection reagent from Promega, Madison, WI, USA; PEI from Sigma; TRIZOL and TPCK-treated trypsin from Thermo Fisher, Waltham, MA, USA; SYBR Green from Bio-Rad, Hercules, CA, USA. Antibodies targeting SARS-CoV-2 Spike (GTX632604) was obtained from Genetex, Irvine, CA, USA. Anti-β-actin antibody (A5316) was purchased from Sigma.
6
SARS-CoV-2 Infectivity Assay Protocol
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Samples quantified by RT-qPCR with a Cq value < 32 were sent to a Biosafety-level-3 lab (Roy Romanow Provincial Laboratory, Saskatchewan Health Authority, Regina, Canada) for infectivity assay. For this, the viral inoculum was added to ready-to-use Vero-76 culture tubes and kept in an incubator at 37 °C and 5% CO2 for 1 h. One mL of RM-02 REFEED medium (Quidel [Diagnostic Hybrids] 10-320500) with 2% FBS, antibiotics and TPCK-treated trypsin (1.0 µg/mL for samples with a Cq value ≤ 25, or 16 µg/mL for samples with Cq value > 25; ThermoFisher Scientific) was then added to each tube and the cells were monitored for CPE (P0d4). On day 4 post-inoculation, 100 µl of the supernatant was removed and added to a new ready-to-use Vero-76 culture tube for the second CPE (P1d4) evaluation. This allowed the virus to replicate in the first assay and increase its infection capability in the second. It is also important for determining that the CPE seen is truly from viral replication and not sample related. This approach is required for samples with low viral load and increases the analytic accuracy.
7
Influenza A Virus Propagation and Titration
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Human alveolar basal epithelial (A549), human embryonic kidney (HEK 293T), and Madin-Darby canine kidney (MDCK) cells were purchased from ATCC. Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) was used to culture and maintain all cell types used.
Influenza A virus strains A/WSN/33 (H1N1), A/HK/1/68 (H3N2), A/HK/54/98 (H1N1), and A/Vietnam/1203/2003 (H5N1) were propagated in MDCK cells supplemented with 0.3% bovine serum albumin (BSA) (Sigma-Aldrich) in the presence of 1 μg/ml tosylphenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Thermo Scientific) and stored as virus stocks at −80 °C until further use. Viral titers were obtained by a plaque assay on MDCK cells. Briefly, serial 10-fold dilutions of virus stocks were adsorbed onto a confluent monolayer of MDCK cells for 1 h at 37 °C. Next, viral inoculums were removed, and cells were washed twice with phosphate-buffered saline (PBS), pH 7.4. Cells were then covered with 1% semisolid agar in DMEM complemented with 0.3% BSA and 1 μg/ml TPCK-treated trypsin. Three days after incubating the plates in an upside down manner, plaques were visualized by staining with crystal violet (84 (link)).
Influenza A virus strains A/WSN/33 (H1N1), A/HK/1/68 (H3N2), A/HK/54/98 (H1N1), and A/Vietnam/1203/2003 (H5N1) were propagated in MDCK cells supplemented with 0.3% bovine serum albumin (BSA) (Sigma-Aldrich) in the presence of 1 μg/ml tosylphenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Thermo Scientific) and stored as virus stocks at −80 °C until further use. Viral titers were obtained by a plaque assay on MDCK cells. Briefly, serial 10-fold dilutions of virus stocks were adsorbed onto a confluent monolayer of MDCK cells for 1 h at 37 °C. Next, viral inoculums were removed, and cells were washed twice with phosphate-buffered saline (PBS), pH 7.4. Cells were then covered with 1% semisolid agar in DMEM complemented with 0.3% BSA and 1 μg/ml TPCK-treated trypsin. Three days after incubating the plates in an upside down manner, plaques were visualized by staining with crystal violet (84 (link)).
8
Antiviral Screening of Influenza Virus Strains
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Antiviral activity was tested against three strains of influenza virus: A/Hamburg/05/2009 (H1N1), A/Hessen/1/2003 (H3N2) and B/Malaysia/2506/2004 (B-Mal). The serine protease inhibitor aprotinin (Carl Roth, Karlsruhe, Germany) and peptides were dissolved in water and stock solutions at a concentration of 10 mM were stored at −20 °C. Viruses were propagated in MDCK II cells in DMEM GlutaMAX supplemented with 1% penicillin/streptomycin, 0.2% bovine serum albumin (BSA, Thermo Fisher Scientific) and 1 µg/mL of TPCK-treated trypsin (Thermo Fisher Scientific). For antiviral screening, MDCK II cells were seeded to 90% confluence in an infection medium (as above, without trypsin) and inoculated with each virus strain at a multiplicity of infection of 1 (MOI = 1). After 1 h, the cells were washed twice with PBS, followed by treatment with each peptide at a 100 µM concentration or aprotinin in the full medium (including trypsin). Cell viability was determined 48 h after treatment using the CellTiterGlo assay, as described above. Virus-treated cells were used as blanks and RLU values were subtracted from the blank before normalizing them to aprotinin, set to one. Triplicate measurements were used to calculate means and standard deviations. Raw data are provided in Supplementary File S3 .
9
Differential Macrophage Infection by Influenza and SARS-CoV-2
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Peripheral blood mononuclear cells were isolated from the peripheral blood of healthy donors under ethics approval No. 2019-1519_1 and differentiated as previously described [15 (link)].
Differentiated macrophages were infected with IAV (PR8) and the SARS-CoV-2 omicron and delta variants. Influenza infection was induced by incubating the cells with virus diluted in DPBS (Thermo Fisher Scientific) supplemented with 0.2% (v/v) human serum albumin (HSA; PAN-Biotech GmbH), 1 mM MgCl2, and 0.9 mM CaCl2 at 37 °C and 5% CO2 for 30 min. Cells were then incubated in RPMI medium supplemented with 1 mM MgCl2, 0.9 mM CaCl2, and 30 ng TPCK-treated trypsin (Thermo Fisher Scientific) for 8 h and 24 h. SARS-CoV-2 stocks were diluted in RPMI medium supplemented with 10% (v/v) HSA. Cells were incubated with inoculum for 60 min, washed once with DPBS, and infected for 8 h and 24 h.
Differentiated macrophages were infected with IAV (PR8) and the SARS-CoV-2 omicron and delta variants. Influenza infection was induced by incubating the cells with virus diluted in DPBS (Thermo Fisher Scientific) supplemented with 0.2% (v/v) human serum albumin (HSA; PAN-Biotech GmbH), 1 mM MgCl2, and 0.9 mM CaCl2 at 37 °C and 5% CO2 for 30 min. Cells were then incubated in RPMI medium supplemented with 1 mM MgCl2, 0.9 mM CaCl2, and 30 ng TPCK-treated trypsin (Thermo Fisher Scientific) for 8 h and 24 h. SARS-CoV-2 stocks were diluted in RPMI medium supplemented with 10% (v/v) HSA. Cells were incubated with inoculum for 60 min, washed once with DPBS, and infected for 8 h and 24 h.
10
Antiviral Activity of Pseudomonas sp. M20A4R8 Extract Against Influenza A Virus
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The bacterial extract was applied before and after IAV inoculation at an MOI of 0.1 in MDCK cells to confirm general antiviral activity, as described elsewhere [8 (link),32 (link)]. Before virus infection, the cells were incubated with serially diluted Pseudomonas sp. M20A4R8 extract (starting from concentrations of 500, 250, 125, 62.5, 31.3, 15.6, 5, 1, and 0.1 µg/mL, diluted with DMEM) at 37 °C for 1 h. Subsequently, the cells were washed with DPBS and infected with IAV (0.1 MOI) at 37 °C for 1 h. Following virus adsorption, the cells were washed and treated with the same concentration of bacterial extract as previously described, and then supplemented with 1 µg/mL of TPCK-treated trypsin (Thermo Fisher Scientific).
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