Trypsin neutralizer solution

Manufactured by Thermo Fisher Scientific

Sourced in United States

Trypsin neutralizer solution is a laboratory product designed to inactivate the proteolytic enzyme trypsin. It is commonly used in cell culture applications to stop the enzymatic action of trypsin during cell detachment and passaging procedures.

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8 protocols using trypsin neutralizer solution

Hydrogel Fabrication and Cell Viability Assay

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HepG2 was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Trypsin-EDTA 0.25%, trypsin neutralizer solution, and Dulbecco’s Modified Eagle Medium (DMEM) were purchased from Gibco (Grand Island, NY, USA). Phosphate buffered saline (PBS), fetal bovine serum (FBS), sodium hydroxide (NaOH), trimethylolpropane triacrylate (TMSPMA), polyethylene glycol diacrylate (PEGDA), and Photo Initiator (PI) were bought from Sigma-Aldrich (St. Louis, MO, USA); and the Live/Dead^® Viability/Cytotoxicity kit from Invitrogen (Waltham, MA, USA).
A polymethyl methacrylate (PMMA) sheet with a thickness of 3 mm and 5 mm was obtained from Fusheng (Taiwan). The cutting laser fiber machine of Laser Top (Vietnam) that has a capacity of 50 W CO₂ laser cutting machine 4060 50W (China) was used. The glasses in the sizes of 24 × 50 mm and 22 × 22 mm were purchased from Jiang Huida Medical Instruments Co., Ltd. (Yancheng, China). Polydimethylsiloxane (PDMS) prepolymer (RTV 615 A) and curing agent (RTV 615 B) were purchased from Momentive Performance Materials (Waterford, NY, USA). The red and blue dyes are food color liquids from Vinh Nam Anh Co., Ltd. (Ho Chi Minh City, Vietnam). ImageJ software was from the National Institute of Health (Bethesda, MD, USA). The OriginPro 2022 software was downloaded from OriginLab (Northampton, MA, USA).

Do T.D., Pham U.T., Nguyen L.P., Nguyen T.M., Bui C.N., Oliver S., Pham P., Tran T.Q., Hoang B.T., Pham M.T., Pham D.T, & Nguyen D.T. (2023). Fabrication of a Low-Cost Microfluidic Device for High-Throughput Drug Testing on Static and Dynamic Cancer Spheroid Culture Models. Diagnostics, 13(8), 1394.

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Expansion and Culture of Melanoma and Melanocyte Cells

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A375 (malignant melanoma) were expanded in high glucose DMEM (DMEM-HG, HyClone), 10% FBS (HyClone), and 1% penicillin/streptomycin (P/S, HyClone). Cells were passaged at ~80% confluence using 0.25% Trypsin-EDTA (HyClone, GE Healthcare). HEM- L158 (HEM, human epidermal melanocytes), were expanded in Medium 254 (M254500, Gibco), 1% human melanocyte growth supplement-2 (S0165, Gibco), and 1% P/S. HEM cells were subcultured at 60–80% confluence using 0.05% Trypsin-EDTA (25300054, Gibco), followed by neutralization with Trypsin Neutralizer Solution (R002100, Gibco). All cells were maintained in humidified incubators at 37°C, 5% CO₂.

Shah M.K., Leary E.A, & Darling E.M. (2018). Integration of hyper-compliant microparticles into a 3D melanoma tumor model. Journal of biomechanics, 82, 46-53.

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Comprehensive Reagent Procurement for Cellular Assays

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DL-SFN, dimethyl sulfoxide (DMSO), propidium iodide (PI), Ponceau S and collagenase were purchased from Sigma-Aldrich. Trypan blue, Dulbecco’s modified Eagle medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, l-glutamine 100×, foetal bovine serum (FBS), penicillin–streptomycin, 0.25% trypsin–EDTA, Medium 254, human melanocyte growth supplement-2 PMA-Free (HMGS-2), RNAse-A, DEPC–water, collagen I bovine, Hank’s balanced salt solution (HBSS), trypsin neutralizer solution and 0.025% trypsin/EDTA solution were obtained from Gibco Life Technologies. Other chemical supplements and compounds were purchased from several companies as follows: CellTiter-Glo luminescent cell viability assay (Promega), turbo DNA free (Ambion, Life Technologies), FITC Annexin V apoptosis detection kit I (BD Pharmingen), TRIzol reagent (Ambion, Life Technologies), methylene blue (Merck), BCA protein assay kit and NP-40 (Pierce, Thermo scientific), ECL Prime Western blotting detection reagents (Amersham), stripping buffer stripAblot and Tween 20 (EuroClone), complete protease inhibitor cocktail (Roche), PageRule prestained protein ladder (Fermentas, part of Thermo Fisher Scientific), superscript III Reverse Transcriptase (Life Technologies) and iQ™ SYBR^® green supermix (Bio-Rad).

Arcidiacono P., Ragonese F., Stabile A., Pistilli A., Kuligina E., Rende M., Bottoni U., Calvieri S., Crisanti A, & Spaccapelo R. (2017). Antitumor activity and expression profiles of genes induced by sulforaphane in human melanoma cells. European Journal of Nutrition, 57(7), 2547-2569.

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Smooth Muscle Cell Growth and Signaling

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Human smooth muscle cell growth medium (M231), smooth muscle growth supplement, trypsin/EDTA solution, trypsin neutralizer solution and HASMCs were obtained from Cascade Biologics (Portland, OR). Fetal bovine serum (FBS), antibiotic-antimycotic mixture, mice TNF-α kits, oligofectamine and 2’,7’-dichlorofluorescein diacetate (DCFDA) were obtained from Life technology (Grand Island, NY). Small interfering RNA (siRNA) oligonucleotides against TNFR 1(sc-29507), p65 (sc-29410), and p47 (sc-76032) and antibodies against human TNFR1 (sc-8436), MSX-2(sc-17729), BMP2(sc-6895), RUNX2(sc-10758), CD68 (sc-9139), β-actin(sc-47778), and anti-hnRNA c1/c2 (sc-32308)were obtained from SantaCruz Biotechnology (Santa Cruz, CA). Antibodies against human NF-kB subunit P65(#558421), NADPH oxidase subunit p47(#610354), and caveoli-1 (610406) were obtained from BDBiosciences (San Jose, CA). Unless otherwise specified, all other chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO).

Lin C.P., Huang P.H., Lai C.F., Chen J.W., Lin S.J, & Chen J.S. (2015). Simvastatin Attenuates Oxidative Stress, NF-κB Activation, and Artery Calcification in LDLR-/- Mice Fed with High Fat Diet via Down-regulation of Tumor Necrosis Factor-α and TNF Receptor 1. PLoS ONE, 10(12), e0143686.

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HASMC Culture and Signaling Pathways

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Human SMC growth medium (M231), smooth muscle growth supplement, trypsin/ethylenediaminetetraacetic acid (EDTA) solution, trypsin neutralizer solution, and HASMCs were obtained from Cascade Biologics (Portland, OR). Fetal bovine serum (FBS), antibiotic–antimycotic mixture, mouse TNF-α kits, oligofectamine, and 2’, 7’-dichlorofluorescein diacetate were obtained from Life Technology (Grand Island, NY). Small interfering RNA (siRNA) oligonucleotides against RAGE and antibodies against human TNFR-1 (sc-8436), MSX2 (sc-17729), BMP-2 (sc-6895), RUNX2 (sc-10758), CD68 (sc-9139), β-actin (sc-47778), and anti-hnRNP c1/c2 (sc-32308) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against human Nox subunit p47 (#610354), and caveoli-1 (#610406) were obtained from BD Biosciences (San Jose, CA). Unless otherwise specified, all other chemicals and reagents obtained from Sigma-Aldrich (St. Louis, MO). All methods in this study were reported in accordance with ARRIVE guidelines.

Lin C.P., Huang P.H., Chen C.Y., Wu M.Y., Chen J.S., Chen J.W, & Lin S.J. (2021). Sitagliptin attenuates arterial calcification by downregulating oxidative stress-induced receptor for advanced glycation end products in LDLR knockout mice. Scientific Reports, 11, 17851.

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Human Arterial Smooth Muscle Cell Assay

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Human SMC growth medium (M231), smooth muscle growth supplement, trypsin/ethylenediaminetetraacetic acid (EDTA) solution, trypsin neutralizer solution, and HASMCs were obtained from Cascade Biologics (Portland, OR). Fetal bovine serum (FBS), antibiotic-antimycotic mixture, mouse TNF-α kits, oligofectamine, and 2',7'-dichloro uorescein diacetate were obtained from Life Technology (Grand Island, NY). Small interfering RNA (siRNA) oligonucleotides against RAGE and antibodies against human TNFR1 (sc-8436), MSX2 (sc-17729), BMP2 (sc-6895), RUNX2 (sc-10758), CD68 (sc-9139), β-actin (sc-47778), and anti-hnRNP c1/c2 (sc-32308) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against human, Nox subunit p47 (#610354), and caveoli-1 (#610406) were obtained from BD Biosciences (San Jose, CA). Unless otherwise speci ed, all other chemicals and reagents obtained from Sigma-Aldrich (St. Louis, MO).

Lin C., Huang P., Chen C., Chen J., Chen J., & Lin S. (2021). Dipeptidyl Peptidase-4 Inhibitor Sitagliptin Attenuates Arterial Calcification By Downregulating Oxidative Stress-Induced Receptor for Advanced Glycation End Products in Low-Density Lipoprotein Receptor Knockout Mice.

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Dissociation of Solid Tumor Samples

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Freshly excised tumors were minced with scalpel blades, and tumor pieces were digested with DMEM/F12 medium containing 5% horse serum, 1 mg/ml collagenase I (Sigma), and 1 mg/ml collagenase D (Sigma) for 2 hr at 37°C. Cells were then washed with Hank’s balanced salt solution (HBSS) (Invitrogen) and suspended in 0.05% Trypsin/EDTA (Invitrogen) for 5 min at room temperature (RT). During trypsinization, cells were passed through 18G, 22G, 27G needles followed by passage through a 40 μm cell strainer (BD Bioscience). Following addition of Trypsin Neutralizer Solution (Invitrogen), cells were collected by brief centrifugation. Cell pellets were washed with HBSS, suspended in DMEM/F12 medium, and analyzed by flow cytometry or FACS sorted.

Tang B., Raviv A., Esposito D., Flanders K.C., Daniel C., Nghiem B.T., Garfield S., Lim L., Mannan P., Robles A.I., Smith WI J.r., Zimmerberg J., Ravin R, & Wakefield L.M. (2014). A Flexible Reporter System for Direct Observation and Isolation of Cancer Stem Cells. Stem Cell Reports, 4(1), 155-169.

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Culturing and Passaging HUVEC Cells

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HUVEC cell line C-003-5C (Invitrogen, USA) were maintained in round 22.1cm² fibronectin (10ng/mL, Invitrogen, USA) coated tissue culture dishes at a concentration of 1.25 x 10⁴cells/mL with a volume of 5 ml per culture dish. The cells were cultured in Medium 200 supplemented with Low Serum Growth Supplement (LSGS, Invitrogen) containing foetal bovine serum (FBS), hydrocortisone, human epidermal growth factor, basic fibroblast growth factor and heparin in a humidified 37°C, 5% CO₂/ 95% air cell culture incubator. We cultured the HUVEC in the flasks for approximately 3 days until the confluency was estimated to be 80%. Sub-culturing was done with Trypsin/EDTA (0.025% Trypsin / 0.01% EDTA solution) at room temperature for 3 minutes whereafter the action of Trypsin/EDTA was blocked by 3 mL of Trypsin Neutralizer solution (Invitrogen, USA). Only 4th-passaged HUVEC were used for all experiments. HUVEC were used, since it is a major endothelial cell type that produces ADAMTS13 in humans and it is readily available and the results are comparable 5 (link), 12 (link)-16 (link). Human umbilical vein endothelial cells are also capable of expressing VWF and also carry ADAMTS13, which makes it a viable option for VWF research 17 (link), 18 (link).

Meiring M., Allers W, & Le Roux E. (2016). Tissue factor: A potent stimulator of Von Willebrand factor synthesis by human umbilical vein endothelial cells. International Journal of Medical Sciences, 13(10), 759-764.

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