MDCK (2.5 × 105) or A549 (5 × 105) cells were seeded in a six-well plate together with control or NXT1 siRNA (1.5 µL of 10 nM siRNA and 7.5 µL of RNAiMax in 500 µL of Opti-MEM) and cultured for 24 h or 48 h. The cells were infected with influenza A/WSN/33 virus at a multiplicity of infection (MOI) = 0.0001, as described previously [14 (link)]. The infected MDCK cells were cultured in DMEM supplemented with 10% FBS, with 1 µg/ml of tosylsulfonyl phenylalanyl chloromethyl ketone-treated trypsin (TPCK-trypsin; Worthington Biochemical Corporation, Lakewood, NJ, USA) to enhance viral replication efficiency while the infected A549 cells were cultured without TPCK-trypsin since the cells are sensitive to trypsin. Culture medium containing virus was collected at the indicated time points (see Figure 2 ) and titrated for virus concentration by plaque assay, as described previously [14 (link)]. In order to check viral protein expressions, the 24 h NXT1-knockdown MDCK cells at 18 h post-infection (hpi) and A549 cells at 48 hpi were collected, lysed with NET buffer (10 mM Tris-HCl/pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% NP-40) plus protease inhibitor cocktail, and boiled with SDS sample buffer for WB analysis of viral and cellular proteins (see Figure 2 ).
Tpck trypsin
Manufactured by Worthington
Sourced in United States
TPCK-trypsin is a laboratory reagent used for protein digestion. It is a form of the enzyme trypsin that has been modified with the chemical reagent TPCK (tosyl-L-phenylalanine chloromethyl ketone) to enhance its specificity and stability. TPCK-trypsin is commonly used in various biochemical and proteomic applications that require controlled protein cleavage.
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Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (7 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Ze5 cytometer, by Bio-Rad (3 mentions)
Mdck cells, by American Type Culture Collection (3 mentions)
Ze5 facs, by Bio-Rad (3 mentions)
Hepes, by Worthington (3 mentions)
Hepes, by Thermo Fisher Scientific (3 mentions)
96 well round bottom plate, by Corning (3 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Alexa fluor647 labeled goat anti human igg secondary ab, by Jackson ImmunoResearch (2 mentions)
Prism v9, by GraphPad (2 mentions)
Opti mem, by Worthington (2 mentions)
Antibiotic antimycotic solution, by Merck Group (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Pen strep, by Merck Group (2 mentions)
Igepal ca 630, by Merck Group (2 mentions)
Gentamicin, by Worthington (2 mentions)
63 protocols using tpck trypsin
1
Influenza Virus Infection and NXT1 Knockdown in MDCK and A549 Cells
2
Reverse Genetics for Influenza A Virus Generation
3
Comprehensive Protein Extraction and Digestion
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0.1 mg from each tissue specimen were cut into small pieces, mixed with lysis buffer (200 mM HEPES, pH 7.5 (AppliChem, Germany), 1% acid-labile surfactant (ALS, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl) methoxy]-1-propanesulfonate)), and incubated at 90 °C for 10 min with mild agitation (500 rpm). Subsequently, samples were further homogenized using a PreCellys Lysis Kit (Bertin instruments, USA) for three cycles (each 30 secundum, at 6500 rpm) and sonicated with a Biorupture device for 20 cycles (each 30 secundum ON and 30 secundum OFF). After centrifugation (at 4 °C, for 10 min at max speed), the supernatant was collected, the pH value was adjusted to pH 8.0 and samples were reduced (5 mM TCEP (Sigma Aldrich, Switzerland), 10 min at 95 °C) and alkylated (10 mM iodacetamide (Sigma Aldrich, USA) in darkness at room temperature for 30 min). A double digest with Lysyl Endopeptidase (Wako, Japan) in a ratio of 1:100 for 2 h, at 37 °C, at 500 rpm and trypsin TPCK treated (Worthington, USA) in a ratio of 1:50 overnight at 37 °C, at 500 rpm was performed.
4
Influenza B Virus Propagation in MDCK Cells
5
Reverse Genetics for Influenza A Virus
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Reverse genetics was performed using pPol I plasmids containing cDNA sequences of the A/WSN/1933 (WSN; H1N1) viral genes between the human Pol I promoter and mouse Pol I terminator as described previously (47 (link)). Briefly, eight pPol I plasmids and pCAGGS protein-expression plasmids for PB2, PB1, PA, and NP were mixed with TransIT-293 (Mirus Bio, Madison, WI) and added to HEK293T cells. At 48 h posttransfection, the cells were treated with 1 μg/mL TPCK-trypsin (Worthington Biochemical, Lakewood, OH) for 30 min and centrifuged at 1,750 × g for 15 min at 4°C, and the supernatant was collected and stored at −80°C. PB2-FLAG virus was generated by replacing pPol I/PB2 wt with pPol I/PB2-FLAG plasmid. For subsequent viral amplification, MDCK cells were infected at an MOI of 10−5, followed by incubation for 2 days in BSA/MEM containing 1 μg/mL TPCK-trypsin.
6
Influenza A virus infection
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Cells were washed with EMEM infection medium [EMEM (ATCC), 10 mM HEPES (Gibco), 0.125% BSA (Gibco), 0.5 µg/mL TPCK trypsin (Worthington Biomedical Corporation)] and incubated with A/WSN/33 for 1.5 h. Cells were then washed with EMEM infection medium and incubated in the same medium throughout the experiment. MOIs are indicated in the figures and/or legends.
7
Influenza Virus Propagation and Quantification
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Biological influenza virus A/California/07/2009 (H1N1pdm09) and recombinant influenza virus A/Perth/16/2009 (H3N2) were used in this study as previously described (28 (link)). Virus propagation was conducted by growing 1:50,000 CP1 virus stocks on confluent Madin-Darby canine kidney cells, kindly provided by Dr. Kanta Subbarao, in infection medium [Eagles’ Minimum Essential Medium (MEM) with L-glutamine (Lonza, Cat. No. BE12-611F) containing 2× antibiotic-antimycotic (Gibco, Cat. No. 15240062), L-glutamine (Lonza, Cat. No. BE17-605E), and TPCK trypsin (Worthington-Biochem, Cat. No. LS003750)] at 37°C. Virus was harvested after significant cytopathic effect was observed. Cellular material was removed through centrifugation at 2,000× g for 10 min at 4°C. The H3N2 virus stock was concentrated through a 30% sucrose cushion to increase the detectable range of virus decay in droplet experiments by ultracentrifugation at 24,000× rpm for 1.5 hours at 4°C after propagated virus was harvested from cells. Following ultracentrifugation, H3N2 virus was resuspended in infection medium and vortexed vigorously. Virus stocks were stored in aliquots at −80°C until use. Infectious virus concentrations were quantified using the tissue culture infectious dose 50 (TCID50) Spearman Karber method, as previously described (53 (link)).
8
Virus Titration in A549 and MDCK Cells
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Triplicate A549 cells and MDCK cells (105 cells per well) were infected with different viruses at a multiplicity of infection (MOI) of 0.01 and incubated at 37°C for 1 h. Cells were then washed with PBS and further incubated with culture medium (supplemented with 2 μg/ml TPCK-Trypsin [Worthington, United States]). Supernatants were collected at 24, 48, and 72 h and subjected to virus tittering by TCID50. Virus titer was determined by end point titration in MDCK in 96-well plates (de Wit et al., 2004 (link)). Briefly, 10-fold serial dilution were conducted and 100 μl/well of virus was added to confluent MDCK cells, plates were then incubated at 37°C, 5% CO2 for 72 h. Each sample consisted of four replicates, and TCID50 was analyzed in using hemagglutinating of chicken RBCs, following the Reed and Muench method (Reed and Muench, 1938 ).
9
Kinetic Analysis of SARS-CoV-2 Spike Binding
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CHO cells stably expressing the prototypic SARS-CoV-2 Spike protein were harvested, washed in wash buffer (PBS 1% BSA 2 mM EDTA) and resuspended in PBS. Cells were then counted and 90’000 cells/well were dispensed into a round-bottom 96 well plate (Corning) to be treated with 10 ug/ml TPCK-Trypsin (Worthington Biochem) for 30 min at 37°C. After a washing step, cells were incubated with 15 ug/ml mAbs solution for 180, 120, 60, 30 or 5 min at 37°C. After the incubation for the allotted time, cells were washed with ice-cold wash buffer and stained with 1.5 ug/ml Alexa Fluor647-labeled Goat Anti-Human IgG secondary Ab (Jackson Immunoresearch) for 30 min on ice in the dark. Cells were then washed twice with cold wash buffer and analyzed using a ZE5 cytometer (Biorad) with acquisition chamber T= 4°C. Binding at each time point (MFI) was determined normalizing to the MFI at 5 minutes time point and data plotted using GraphPad Prism v. 9.1.1
10
Quantifying H9N2 Virus Titers in MDCK Cells
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Plaque assay performed on MDCK cells was used to detect virus titers as described previously (Tobita et al., 1975 (link)). Briefly, MDCK cells were seeded into 6-well plates. Confluent monolayers were washed with PBS and infected with H9N2 virus or cell culture supernatants. The inoculum was discarded after incubation for 1 h and the remaining cells were washed with PBS. An overlay consisting of a mixture of aga-rose 1.6% (Lonza, USA) and double-strength DMEM with TPCK-trypsin 0.25 μg/ml (Worthington, USA) was added to the above cells and incubated at 37°C for 72 h. Plaques were stained with crystal violet 0.1% and counted.
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