Acetylated trypsin

Manufactured by Merck Group

Sourced in United States, Japan

Acetylated trypsin is a laboratory reagent used for the hydrolysis of peptide bonds in proteins. It is a modified form of the enzyme trypsin, with the addition of acetyl groups to the enzyme structure. This modification alters the enzyme's activity and specificity, making it useful for various applications in proteomics and protein chemistry.

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Lab products found in correlation

Bacto agar, by BD (4 mentions) Horseradish peroxidase conjugated goat anti mouse antibody, by Agilent Technologies (2 mentions) Dmem f12, by Merck Group (2 mentions) Crystal violet, by Nacalai Tesque (2 mentions) Minimum essential medium (mem), by Merck Group (1 mentions) Gentamicin, by Merck Group (1 mentions) Vero e6 cell, by American Type Culture Collection (1 mentions) Vero e6 cells, by BEI Resources (1 mentions) Vero e6 tmprss2 t2a ace2 cells, by BEI Resources (1 mentions) 3 amino 9 ethylcarbazole aec, by Merck Group (1 mentions) Saponin, by Merck Group (1 mentions) Neutral red, by Merck Group (1 mentions) Seakem me agarose, by Lonza (1 mentions) Paraformaldehyde (pfa), by Polysciences (1 mentions) Hb 65, by American Type Culture Collection (1 mentions) Lead stain solution, by Merck Group (1 mentions) Em 14830ruby2, by JEOL (1 mentions) Jem 1400plus, by JEOL (1 mentions) Quetol 812, by Nisshin EM (1 mentions) Gentamicin sulfate solution, by Merck Group (1 mentions)

30 protocols using acetylated trypsin

Influenza A Virus Propagation in MDCK Cells

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Madin–Darby canine kidney (MDCK) cells were cultured in minimum essential medium (MEM; Sigma-Aldrich Co. LLC, St. Louis, MO) supplemented with 5% (v/v) heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich Co.) and 50 µg/mL gentamicin at 37 °C in humidified air containing 5% CO₂. Influenza A virus strains [A/Puerto Rico/8/34 (H1N1)] and [A/MEMphis/1/1971 (H3N2)] were propagated at 37 °C using MDCK cells in serum-free medium (SFM; Thermo Fisher Scientific Japan K.K., Kanagawa, Japan) supplemented with 2 µg/mL acetylated trypsin (Sigma-Aldrich Co.) and 50 µg/mL gentamicin.

Kobayashi K., Shono C., Mori T., Kitazawa H., Ota N., Kurebayashi Y, & Suzuki T. (2022). Protein-bound sialic acid in saliva contributes directly to salivary anti-influenza virus activity. Scientific Reports, 12, 6636.

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Serum-Neutralizing Antibody Titer Assay

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Serum-neutralizing antibody titers in mice were measured using methods described in previous research [6 (link)]. Briefly, mouse serum samples were heat-inactivated at 56 °C for 30 min and mixed with 100-fold median tissue culture infectious dose (TCID₅₀) of MO/15 or Swan/Hok virus and incubated for 1 h at room temperature. The mixture was inoculated onto confluent monolayers of MDCK cells and incubated at 35 °C for 1 h. Unbound viruses were removed, and cells were washed with PBS. Cells were incubated with MEM containing 5 μg/mL acetylated trypsin (Sigma-Aldrich, St. Louis, MO, USA). Cytopathic effects were observed after 72 h of incubation, and the neutralizing antibody titers were determined as the reciprocal of the serum dilution yielding 50% inhibition of the cytopathic effects.

Hayashi H., Isoda N., Bazarragchaa E., Nomura N., Matsuno K., Okamatsu M., Kida H, & Sakoda Y. (2020). Potency of an Inactivated Influenza Vaccine against a Challenge with A/Swine/Missouri/A01727926/2015 (H4N6) in Mice for Pandemic Preparedness. Vaccines, 8(4), 768.

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Expansion and Maintenance of SARS-CoV-2 and Influenza Strains

Cited 2 times

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Vero E6 cells (ATCC, Cat#CRL-1586, Manassas, VA, USA) were maintained in Modified Eagle’s Medium (MEM) supplemented with 10% fetal bovine serum (FBS). Human ACE2-overexpressing A549 cells (ACE2-A549), a gift from the tenOever laboratory [10 (link)], were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS. The following SARS-CoV-2 strains were expanded in Vero E6 cells: infectious-clone-derived mNeonGreen SARS-CoV-2 (icSARS-CoV-2 mNG), a kind gift from Dr. Pei-Yong Shi [11 (link)]; USA-WA1/2020 (WA/01, BEI Resources Cat #NR-52281, Manassas, VA, USA); and MA10 (BEI Resources Cat #NR-55329). SARS-CoV-2 strain B.1.1.529 omicron (BEI Resources Cat #NR-56461) was expanded in Vero E6-TMPRSS2-T2A-ACE2 cells (BEI Resources Cat #NR-54970). Influenza A/Hong Kong/8/68 (H3N2) was originally from Charles River Laboratories (Wilmington, MA, USA) and subsequently grown in Madin–Darby canine kidney (MDCK) cells (ATCC Cat #NBL-2) in MEM supplemented with 1% bovine serum albumin and 10 μg/mL acetylated trypsin (Sigma, St. Louis, MO, USA).

Lim S.Y., Guo Z., Liu P., McKay L.G., Storm N., Griffiths A., Qu M.D., Finberg R.W., Somasundaran M, & Wang J.P. (2022). Anti-SARS-CoV-2 Activity of Adamantanes In Vitro and in Animal Models of Infection. COVID, 2(11), 1551-1563.

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Influenza Virus Propagation and Titration

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The viruses used are summarized in Table 1. All IAV were propagated in 10-day-old embryonated specific-pathogen-free (SPF) chicken eggs incubated for 2 days at 37°C. IAV were titrated on confluent MDCK cells in presence of 1 μg/ml of acetylated trypsin (Sigma-Aldrich). After 24 h, cells were fed by addition of new medium, and 48 h post-infection (p.i.), the cells were fixed with 4% paraformaldehyde (Polysciences, Warrington, PA, USA). The plates were stained with anti-NP antibody (HB-65, ATCC) in saponin (Sigma-Aldrich), followed by horseradish peroxidase-conjugated goat anti-mouse antibody (Dako, Baar, Switzerland), and a final color reaction with 3-amino-9-ethylcarbazole (AEC, Sigma-Aldrich). Titers were calculated using the Reed and Muench formula.

Ricklin M.E., Vielle N.J., Python S., Brechbühl D., Zumkehr B., Posthaus H., Zimmer G, & Summerfield A. (2016). Partial Protection against Porcine Influenza A Virus by a Hemagglutinin-Expressing Virus Replicon Particle Vaccine in the Absence of Neutralizing Antibodies. Frontiers in Immunology, 7, 253.

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Viral Infection Kinetics in Cell and Egg Models

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DF-1 and Calu-3 cells (90% confluent in collagen-coated 24-well plates) were infected in triplicate with the indicated viruses at an MOI of 0.005 or 0.03, respectively. After 1 h at 37°C, the DF-1 and Calu-3 cells were washed with PBS and maintained in Dulbecco modified Eagle medium–Ham’s F-12 medium (DMEM-F12) containing 0.2% bovine serum albumin (BSA) and 2 μg acetylated trypsin/ml (Sigma-Aldrich) at 33°C or 37°C, respectively. The supernatants were collected at the times postinfection indicated throughout the text above. Nine-day-old embryonated chicken eggs (5 eggs per group) were inoculated with 1 × 10⁵ FFU of the viruses described above, and allantoic fluids were collected at the times postinoculation indicated throughout the text above. Virus titers were determined by FFU assays on MDCK cells as described above.

Arai Y., Elgendy E.M., Daidoji T., Ibrahim M.S., Ono T., Sriwilaijaroen N., Suzuki Y., Nakaya T., Matsumoto K, & Watanabe Y. (2020). H9N2 Influenza Virus Infections in Human Cells Require a Balance between Neuraminidase Sialidase Activity and Hemagglutinin Receptor Affinity. Journal of Virology, 94(18), e01210-20.

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Viral Infection and Titer Quantification

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DF-1 and Calu-3 cells (90% confluent in collagen-coated 24-well plates) were infected in triplicate with the indicated viruses at an MOI of 0.005 and 0.001, respectively. After 1 h at 37°C, the cells were washed with PBS and maintained in DMEM-F12 containing 0.2% BSA and 2 μg acetylated trypsin/ml (Sigma-Aldrich) at 33°C or 37°C. The supernatants were collected at the indicated times post-infection and viral titers were determined by assays of focus-forming units (FFU) as described previously [47 (link)].

Arai Y., Kawashita N., Ibrahim M.S., Elgendy E.M., Daidoji T., Ono T., Takagi T., Nakaya T., Matsumoto K, & Watanabe Y. (2019). PB2 mutations arising during H9N2 influenza evolution in the Middle East confer enhanced replication and growth in mammals. PLoS Pathogens, 15(7), e1007919.

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MDCK Cell-Based Virus Titration

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Confluent monolayers of MDCK cells in 96-well plates were inoculated with serial half log dilutions of virus samples and incubated for 6 days at 34 °C in 5% CO₂ in the presence of 10 μg/mL acetylated trypsin (Sigma). Virus titers were calculated for each sample using the Reed-Muench method [21] , based on the CPE in individual wells observed under an inverted microscope.

Sato K., Takahashi Y., Adachi Y., Asanuma H., Ato M., Tashiro M, & Itamura S. (2019). Efficient protection of mice from influenza A/H1N1pdm09 virus challenge infection via high avidity serum antibodies induced by booster immunizations with inactivated whole virus vaccine. Heliyon, 5(1), e01113.

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Plaque Assay for Viral Quantification

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Plaque assays were performed as described previously [29 (link)]. Briefly, the mouse lung homogenates were diluted in MEM without FCS, applied in tenfold dilutions onto confluent monolayers of MDCK cells, and incubated at 35 °C with 5% CO₂. After 1 h, the unbound virus was removed by discarding the supernatant and washing the cells with PBS. The cells were then overlaid with MEM containing 5 µg/mL acetylated trypsin (Sigma-Aldrich) and 1% Bacto Agar (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). After incubation for 48 h at 35 °C, the cells were stained with 0.005% neutral red. After incubation for another 24 h at 35 °C, the number of plaques was counted. The number of plaque-forming units (PFU) was calculated as the product of the reciprocal value of the highest virus dilution and the number of plaques in the dilution.

Spruit C.M., Nemanichvili N., Okamatsu M., Takematsu H., Boons G.J, & de Vries R.P. (2021). N-Glycolylneuraminic Acid in Animal Models for Human Influenza A Virus. Viruses, 13(5), 815.

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Serum Neutralization Assay for H2N3 Virus

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Serum neutralizing antibody titers were measured according to the method of Sakabe et al [25 (link)]. Briefly, test sera and 100 TCID₅₀ of A/swine/Missouri/2124514/2006
(H2N3) or vaccine strain virus were mixed and incubated for 1 hr at room temperature. The mixture was inoculated onto MDCK cells and incubated at 35°C for 1 hr. Unbound viruses were removed and the cells were washed with PBS. The
cells were subsequently incubated in MEM containing 5 µg/ml acetylated trypsin (Sigma-Aldrich). Cytopathic effects were observed after 72 hr incubation and neutralizing antibody titers were
determined as the reciprocal of the serum dilution yielding 50% inhibition of the cytopathic effects.

SUZUKI M., OKAMATSU M., HIONO T., MATSUNO K, & SAKODA Y. (2017). Potency of an inactivated influenza vaccine prepared from A/duck/Hokkaido/162/2013 (H2N1) against a challenge with A/swine/Missouri/2124514/2006 (H2N3) in mice. The Journal of Veterinary Medical Science, 79(11), 1815-1821.

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Viral Titers in Lung Tissue

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Live lung viral titers were determined by plaque assay using Madin Darby canine kidney (MDCK) cells (provided by Dr. Adam Lauring, University of Michigan). MDCK cells were cultured in 6 well plates overnight until the cell monolayer reached >70% confluence. Lung supernatants were thawed at room temperature and diluted to different dilutions (i.e., 10⁻¹-10⁻⁶) with 0.1% BSA in 1x PBS. Cells were washed twice with 1x PBS followed by 1h incubation with 400 μl lung supernatants at 37°C with shaking every 15 min. Cells were washed twice with 1x PBS to remove excess virus. The cells were then covered with 2 ml over-lay medium containing a 1:1 mixture of 2% agarose and 2x DMEM medium containing 2 μg/ml (final concentration) acetylated trypsin (Sigma-Aldrich). Plates were incubated at 37°C for 72 h. The agarose was removed from the plates and the cells stained with 0.3% crystal violet for 5 min. The plates were washed and viral plaques were counted. The plaque forming units per gram (pfu/g) were calculated using the formula: (# plaques x dilution factor)/weight of lung lobe.

Kulkarni U., Zemans R.L., Smith C.A., Wood S., Deng J.C, & Goldstein D.R. (2019). Excessive neutrophil levels in the lung underlie the age-associated increase in influenza mortality. Mucosal immunology, 12(2), 545-554.

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