Tryptase

Manufactured by Merck Group

Sourced in United States, China

Tryptase is a laboratory equipment product manufactured by Merck Group. Tryptase is a serine protease enzyme that is primarily found in the secretory granules of mast cells. It plays a role in the body's inflammatory response and can be used for research purposes in the laboratory setting. Detailed information about the intended use or interpretation of Tryptase's function is not available in this factual and unbiased description.

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Human lung tryptase (2 citations)

10 protocols using tryptase

Fibroblast-Mast Cell Interaction in IPF

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Primary lung fibroblasts from healthy individuals or patients with IPF in monoculture or in co-culture with LAD2 in 6-well plates were cultured for 72 h, followed by 24 h of starvation in DMEM containing 0.4% FCIII before addition of fresh DMEM containing 0.4% FCIII with or without chymase (1 ng/mL), tryptase (75 ng/mL) or a combination of chymase (1 ng/mL) and tryptase (75 ng/mL) (both from Sigma-Aldrich, St Louis, MO, USA). The chosen concentrations of chymase and tryptase had previously been evaluated [19 (link)]. All incubations were made at 37 °C with 5% CO₂. Cell supernatants were collected after 72 h and stored at −20 °C.

Bagher M., Rosmark O., Elowsson Rendin L., Nybom A., Wasserstrom S., Müller C., Zhou X.H., Dellgren G., Hallgren O., Bjermer L., Larsson-Callerfelt A.K, & Westergren-Thorsson G. (2021). Crosstalk between Mast Cells and Lung Fibroblasts Is Modified by Alveolar Extracellular Matrix and Influences Epithelial Migration. International Journal of Molecular Sciences, 22(2), 506.

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Isolation and Culture of Human Uterosacral Ligament Fibroblasts

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Fresh sterile uterosacral ligament tissues, measuring approximately 0.5×0.5×0.5 cm were collected and then cut into pieces with a diameter <0.1cm for primary culture [25 ]. After collection, tissues were immediately washed with phosphate-buffered saline (PBS). The human uterosacral ligament fibroblasts (hUSLFs) were isolated following digestion with type I collagenase (Solarbio Science & Technology Co., Ltd., Beijing, China) and 0.25% tryptase (Sigma-Aldrich, St. Louis MO, USA). After tissue digestion, the medium was centrifuged at 1500 rpm for 5 minutes. The isolated cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Thermofisher Scientific, Waltham, MA, USA) containing 20% fetal bovine serum (FBS) and 1% antibiotics. The hUSLFs at 3–8 generations of exponential growth phase were selected for the subsequent experiments.

Zhao X., Liu L., Li R., Wei X., Luan W., Liu P, & Zhao J. (2018). Hypoxia-Inducible Factor 1-α (HIF-1α) Induces Apoptosis of Human Uterosacral Ligament Fibroblasts Through the Death Receptor and Mitochondrial Pathways. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 24, 8722-8733.

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Investigating PAR-2 Signaling Pathway

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Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS) and 0.25% Trypsin–EDTA solution were purchased from Gibco-BRL (Grand Island, NY, USA). Tryptase was purchased from Sigma-Aldrich (St. Louis, MO, USA), and it is the human lung Tryptase, which is a neutral serine protease and the predominant protein in mast cell granules. PAR-2 inhibitor FSLLRY-NH2 (FS) was synthesised by CL Bio-Scientific Inc. (Xi An, China). CCK-8, RIPA buffer and the BCA kit were purchased from Beyotime (Shanghai, China). Rabbit anti-PAR-2 polyclonal antibody and fluoroshield mounting medium with 4′,6-diami-dino-2-phenylindole (DAPI) were purchased from Abcam (Hongkong, China). Anti-TLR4 monoclonal antibody, anti-VCAM-1 antibody (EPR5 047) and anti-occludin antibody (EPR8208) were purchased from Abcam (Hongkong, China). Anti-GAPDH antibody was purchased from Bioworld Technology, Inc. (USA). Anti-p44/42 MAPK monoclonal antibody (extracellular regulated protein kinases, ERK), anti-Phospho-p44/42 monoclonal antibody (phosphoERK) and NF-kappa B were purchased from Cell Signaling (Beverly, MA, USA).Anti-rabbit and anti-mouse secondary antibodies were all purchased from Jackson Immuno Research Laboratories Inc. (Boston, MA, USA).

Zhou Q., Wang Y.W., Ni P.F., Chen Y.N., Dong H.Q, & Qian Y.N. (2018). Effect of tryptase on mouse brain microvascular endothelial cells via protease-activated receptor 2. Journal of Neuroinflammation, 15, 248.

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DDX46 Expression and Silencing in Cells

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DMEM and FBS were purchased from Santa Co., Ltd. DMSO, crystal violet and tryptase were purchased from Sigma-Aldrich (Merck KGaA). Cell Counting Kit-8 (CCK-8) kits, Annexin V/PI cell apoptosis detection kits and TaqMan™ reverse transcription reagents (cat. no. 4304134) were obtained from Santa Cruz Biotechnology, Inc. TRIzol^® kit, the transfection reagent Lipofectamine 3000^® and 3,3′-diaminobenzidine (DAB) reagent kit were provided by Thermo Fisher Scientific, Inc. Rabbit anti-DDX46 (1:500; cat. no. ab72083) antibody was purchased from Abcam. The forward (F) and reverse (R) primers sequences for RT-qPCR of DDX46 were designed and synthesized by Arraystar, Inc., and were as follows: DDX46 F, 5′-AAAATGGCGAGAAGAGCAACG-3′ and R, 5′-CATCATCGTCCTCTAAACTCCAC-3′; GAPDH F, 5′-TGACTTCAACAGCGACACCCA-3′ and R, CACCCTGTTGCTGTAGCCAAA-3′. The preparation and packaging of DDX46 gene RNAi target sequence and shDX46 lentivirus (shDDX46) were designed and synthesized by Arraystar, Inc.

Lin Q., Jin H.J., Zhang D, & Gao L. (2020). DDX46 silencing inhibits cell proliferation by activating apoptosis and autophagy in cutaneous squamous cell carcinoma. Molecular Medicine Reports, 22(5), 4236-4242.

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Calpain I Inhibitor Assay Protocol

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Calpain I (isolated form human erythrocytes), kaolin, carrageenan,
cremophor, dimethyl sulphoxide (DMSO), tryptase (from human lung) and
urethane were obtained from Sigma-Aldrich (St. Louis, MO, USA). E-64c
(2S,3S)-3-[[(2S)-4-methyl-1–(3-methylbutylamino)-1-oxopentan-2-yl]carbamoyl]oxirane-2-carboxylic
acid) was purchased from ApexBio (Huston, Texas, USA) and E-64d
(2S,3S)-trans-Epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester)
was purchased from AdooQ Bioscience (Irvine, CA, USA). E-64c and E-64d
were prepared in vehicle (1:1:8; DMSO: cremophor: saline) on the day
of use. Isoflurane and euthansol were purchased from CDMV (Dartmouth,
NS, Canada).

McDougall J.J., McConnell M, & Reid A.R. (2021). Intracellular versus extracellular inhibition of calpain I causes differential effects on pain in a rat model of joint inflammation. Molecular Pain, 17, 17448069211016141.

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Effect of Glycosaminoglycans on Corin Proteolysis

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HEK293 cells expressing WT corin were cultured in 6-well plates at 37°C in the presence of monensin (2 µM). After 48 h, the cells were washed with PBS. The conditioned medium containing recombinant PCSK6 was added to the cell culture together with heparan sulfate (Sigma H7640, 5 and 50 µg/mL) or chondroitin sulfate (Sigma C4384, 5 and 50 µg/mL). After incubation at 37°C for 2 h, the cells were washed and lysed. Corin protein fragments in the cell lysates were examined by SDS-PAGE and Western blotting. To verify the effects of heparan and chondroitin on protease activity, a tryptase assay was performed (Anower et al. 2013 (link)), in which 5 µg/mL of haparan sulfate or chondroitin sulfate was added to a 100 µL of reaction mixtures with 5 ng of tryptase (Sigma) and 50 nM of chromogenic substrate S-2288 (Chromogenix). The reaction mixtures were incubated at 37°C for different time periods, during which tryptase activity was assayed by monitoring the absorbance at 405 nm in a plate reader. Each data point was assayed in quadruplicate in six experiments.

Chen S., Wang H., Li H., Zhang Y, & Wu Q. (2017). Functional Analysis of Corin Protein Domains Required for PCSK6-mediated Activation. The international journal of biochemistry & cell biology, 94, 31-39.

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Purification and Characterization of Recombinant TTR Variants

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Recombinant TTR variants were expressed and purified as described previously (21 (link)). Human fibrinogen was isolated from citrate-heparin–treated human plasma by affinity chromatography on recombinant clamping factor 221–559 fragment (24 (link)) and was absorbed with lysine-Sepharose 4B and gelatin-Sepharose 4B to remove traces of plasminogen and fibronectin, respectively. Enzymes purchased from Sigma-Aldrich were plasmin (P1867), proteinase K (P2308), chymo Trypsin (C2160000), tPA (T0831), plasminogen (SRP6518), thrombin (T7572), and tryptase (T7063). Trypsin was purchased from Promega (V5280) and recombinant human kallikrein 12 from Biotechne (3095-S.E.). All the enzymes used were able to cleave the C-terminal end of Lys in the d-Val-Leu-Lys 4-nitroanilide dihydrochloride peptide (Sigma-Aldrich, V0882) following the manufacturer's instructions. All other reagents including α2-antiplasmin (SRP6313) were purchased from Sigma-Aldrich unless otherwise stated.

Mangione P.P., Verona G., Corazza A., Marcoux J., Canetti D., Giorgetti S., Raimondi S., Stoppini M., Esposito M., Relini A., Canale C., Valli M., Marchese L., Faravelli G., Obici L., Hawkins P.N., Taylor G.W., Gillmore J.D., Pepys M.B, & Bellotti V. (2018). Plasminogen activation triggers transthyretin amyloidogenesis in vitro. The Journal of Biological Chemistry, 293(37), 14192-14199.

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Investigating Inflammatory Signaling Pathways

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Antibodies of PAR2, STAT3, p53, and a STAT3 shRNA kit were purchased from Santa Cruz Biotech (Shanghai, China). Reagents for quantitative real time RT-PCR (qRT-PCR) and Western blotting were purchased from Invitrogen (Shanghai, China). An annexin V kit and tryptase were purchased from Sigma Aldrich (Shanghai, China). The active PAR2 peptide and control peptide were purchased from Alibaba Biotech (Hangzhou, China).

Luo R., Wang X., Dong Y., Wang L, & Tian C. (2014). Activation of protease-activated receptor 2 reduces glioblastoma cell apoptosis. Journal of Biomedical Science, 21(1), 25.

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Fluorescence Lifetime Imaging of Heparin, Histamine, and Tryptase

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Heparin (Biochrom, Berlin, Germany), histamine (Sigma-Aldrich, St. Louis, MO, USA) and tryptase (Sigma-Aldrich, St. Louis, MO, USA) were stored 2 h at room temperature before measurements. TPE-FLIM parameters τ₁, τ₂ and τ_m were recorded with laser excitation at 760 nm with 100 fs pulses and a repetition rate of 80 MHz at 8 mW.

Kröger M., Scheffel J., Nikolaev V.V., Shirshin E.A., Siebenhaar F., Schleusener J., Lademann J., Maurer M, & Darvin M.E. (2020). In vivo non-invasive staining-free visualization of dermal mast cells in healthy, allergy and mastocytosis humans using two-photon fluorescence lifetime imaging. Scientific Reports, 10, 14930.

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Immunohistochemical Analysis of Dupuytren's Disease

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All Dupuytren’s tissue samples were fixed in formalin, longitudinally bisected, and embedded in paraffin wax, and 7-μm sections from the cut surface were processed for immunohistochemistry (18 (link)). Sequential sections were stained with mouse monoclonal anti–α-SMA antibody (Sigma, A2547), anti-CD68 antibody (Dako, clone K61), and tryptase (Sigma, 342M-17). Antibodies were detected using a two-stage polymer enhancer system (Sigma). Mouse serum at the same protein concentration as the monoclonal antibody solution was used as a control.

Izadi D., Layton T.B., Williams L., McCann F., Cabrita M., Espirito Santo A.I., Xie W., Fritzsche M., Colin-York H., Feldmann M., Midwood K.S, & Nanchahal J. (2019). Identification of TNFR2 and IL-33 as therapeutic targets in localized fibrosis. Science Advances, 5(12), eaay0370.

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