Trypsin solution

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, United Kingdom, Australia

Trypsin solution is a proteolytic enzyme used for cell dissociation and the digestion of proteins. It is commonly used in cell culture and molecular biology applications to release adherent cells from the culture surface and separate them into a single-cell suspension.

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HBSS trypsin–EDTA solution Trypsin–EDTA(1×) solution Trypsin-EDTA (0.05%) solution Trypsin/1 mM EDTA solution Trypsin ethylenediaminetetraacetic acid (EDTA) solution Trypsin-EDTA (0.25%) solution Trypsin—EDTA (1X) solution Trypsin digestive solution Trypsin-EDTA solution Trypsin-ethylenediaminetetraacetic acid solution

210 protocols using trypsin solution

Culturing HEK293T and Primary Rat Neurons

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HEK293T cells were a gift from Dr. Dasaradhi Palakodeti, InStem. HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (#21,331-020 Thermo) containing 10% fetal bovine serum (#F2442 Sigma) at 37 °C and 5% CO 2 . Cells were passaged with 0.25% trypsin solution (#15,090,046 Thermo) for 2 min and neutralized using the same culture medium. Primary neuronal cultures were obtained from E18 rat embryos. Primary cultures 1 3 were processed as previously described [10] . Briefly, cortices from both the hemispheres were dissected out from E18 embryos in ice cold Hank's balanced salt solution (#H6648-1L Sigma). The tissue was incubated with 0.25% trypsin solution for 5 min at 37 °C to enzymatically dissociate the cells and finally manually triturated in Minimum Eagles Medium (#11,095,080 Thermo) containing 10% FBS (#F2442 Sigma) to generate a single cell suspension. Cells were plated at a density of 6 × 10 4 cells/cm 2 on glass coverslips coated with 0.2 mg/ml poly-L-lysine (#P2636-100MG Sigma) made in Borate buffer pH 8.5. Neurons were cultured in MEM medium for 4 h after plating following which they were cultured in neurobasal medium (#21,103-049 Thermo) containing B27 supplement (#17,504-044 Thermo) and 1 × Glutamax (#35,050,061 Thermo) for 11 days at 37 °C and 5% CO 2.

D'Souza M.N., Ramakrishna S., Radhakrishna B.K., Jhaveri V., Ravindran S., Yeramala L., Nair D., Palakodeti D, & Muddashetty R.S. (2022). Function of FMRP Domains in Regulating Distinct Roles of Neuronal Protein Synthesis. Molecular neurobiology, 59(12).

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Isolation and Characterization of Epidermal Stem Cells

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Back skin was excised and adipose tissue was removed. The skin was placed on a layer of trypsin solution (0.25%, Life Technologies Germany) with the epidermal side facing upwards. After incubation for 2 hours, the epidermis was separated from the dermis using blunt forceps. The epidermis was minced with scalpels and digested in 10 ml trypsin solution (0.25%, Life Technologies) for another hour. The digest was stopped by adding an equal volume of medium (DMEM (2/3) + HAM`s F12 (1/3)) supplemented with 10% calcium free FBS and the cells were strained through a 40 μm nylon mesh. Cells were then centrifuged for 8 min at 500 g at RT and the pellet was resuspended in 2 ml FACS buffer. Epidermal stem cell populations were stained according to Jensen et al. [35 (link)]. For flow cytometry, cells were stained using the following antibodies: anti-CD45-FITC (1:400, eBioscience), anti-CD49f-PE (1:200, eBioscience), anti-CD34-eF660 (1:50, eBioscience), anti-Sca1-PerCP-Cy5.5 (1:200, eBioscience), anti-CD117-APC-Cy7 (1:800, Biolegend). Cell sorting and analysis was performed on an ARIA III cell sorter (BD) and data were recorded using DIVA software (BD) and analyzed using FlowJo (FlowJo, LLC).

Hiller B., Hoppe A., Haase C., Hiller C., Schubert N., Müller W., Reijns M.A., Jackson A.P., Kunkel T.A., Wenzel J., Behrendt R, & Roers A. (2018). Ribonucleotide excision repair is essential to prevent squamous cell carcinoma of the skin.. Cancer research, 78(20), 5917-5926.

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Isolation of Neonatal Microglia

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Microglia were prepared from neonatal rat or mouse pups at postnatal day (P) 2–3. The cortices were separated from meninges and minced with a sterile razor blade. Tissue pieces were transferred into 2.5% Trypsin solution (Life Technologies/Gibco) containing DNAse (SIGMA). After incubation at 37 °C for 25 min the Trypsin solution was removed, cortices were washed with 30% FBS in DPBS and serially triturated with 30% FBS in Dulbecco's phosphate-buffered saline (DPBS) (Life Technologies/Gibco) containing DNAse. The cell suspension was gently spun at 200g for 15 min and the pellet resuspended in DMEM (Life Technologies/Gibco), containing 10% FBS (Life Technologies/Gibco), 100 units per millilitre penicillin and 100 mg ml⁻¹ streptomycin (Life Technologies/Gibco). Cells were plated into poly-D-lysine pre-coated T-75 flasks at a density of 2–3 cortices per flask. On day 3 in vitro, fresh medium was added and cells were grown for 1 more day. On day 4 in vitro, flasks were placed onto a shaker platform, preheated to 37 °C and microglia cells were shaken off the cortical cell layer at 200 r.p.m. for 2 h. The medium containing mostly microglia cells was removed from the flasks and cells were spun at 200g for 15 min. The cell pellets were gently resuspended in culture medium and the microglia density was adjusted to 5000 cells per microlitre.

Ryu J.K., Petersen M.A., Murray S.G., Baeten K.M., Meyer-Franke A., Chan J.P., Vagena E., Bedard C., Machado M.R., Coronado P.E., Prod'homme T., Charo I.F., Lassmann H., Degen J.L., Zamvil S.S, & Akassoglou K. (2015). Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation. Nature Communications, 6, 8164.

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Quantifying Collagen Production in Osteoblasts

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Total collagen production by pre-osteoblasts growing on functionalized 3D scaffolds was detected by the Sirius red staining method.^{61 (link)–62 (link)} Briefly, MC3T3 cells after 1 week of growth on 3D scaffolds were fixed by 4% PFA solution and stained by Sirius red stain solution (0.1% Direct Red 80 in saturated picric acid) for 16 hours. Stained 3D scaffolds were washed by DI water and dehydrated by 100% ethanol. After drying, the stain on 3D scaffolds was eluted by 0.2 M NaOH: methanol (v/v, 1: 1) solution for 15 min and absorbance was measured by UV-vis absorbance microplate reader at 490 nm. To normalize the collagen amount to cell numbers, another series of scaffolds were seeded with the same density of cells and cultured for 1 week under the same condition. Cells on the scaffolds were then trypsinized using 0.5% trypsin solution (Thermo Fisher Scientific, Waltham, MA) and counted under microscope. The relative collagen amount was calculated by normalizing the obtained OD value to the number of cells observed on each type of scaffold.

Liu X., Miller AL I.I., Park S., George M.N., Waletzki B.E., Xu H., Terzic A, & Lu L. (2019). Two-Dimensional Black Phosphorus and Graphene Oxide Nanosheets Synergistically Enhance Cell Proliferation and Osteogenesis on 3D-Printed Scaffolds. ACS applied materials & interfaces, 11(26), 23558-23572.

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Culturing U373MG-CD14 Glioblastoma Cells

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The human brain glioblastoma cancer cell line (U373MG-CD14) cells were obtained from Dr Michael Carty (Trinity College Dublin). Cells were cultured in Dulbecco’s Modified Eagle’s Medium-high glucose (Sigma-Aldrich) supplemented with 10% foetal bovine serum (Sigma-Aldrich) and 1% penicillin and streptomycin mixture (Thermo Fisher Scientific) in TC flask T25, standard for adherent cells (Sarstedt). The cultures were maintained under a condition of 5% (v/v) CO2 and 37 °C in a humidified incubator. Culture medium was changed every 2 days until reaching around 80% confluency. Cells were then brought into suspension using 0.25% trypsin solution (Thermo Fisher Scientific) and subcultured in new flasks.

He Z., Liu K., Manaloto E., Casey A., Cribaro G.P., Byrne H.J., Tian F., Barcia C., Conway G.E., Cullen P.J, & Curtin J.F. (2018). Cold Atmospheric Plasma Induces ATP-Dependent Endocytosis of Nanoparticles and Synergistic U373MG Cancer Cell Death. Scientific Reports, 8, 5298.

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Enzymatic Extraction of Protein-Enriched Compounds

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Jelly fig (Ficus awkeotsang Makino) and, pea (Pisum sativum L.) for PE extraction, were purchased from a local market in Taipei, Taiwan. Soy protein was from Gemfont (Taipei). Methylene blue and sodium chloride were from Riedel-de Haën (Seelze, Germany). Acetone, methanol, hydrochloride solution and fluorescein isothiocyanate (FITC)-casein were from Merck (Darmstadt, Germany). Trypsin solution was from ThermoFisher Scientific (Waltham, MA, USA). Pancreatic α-amylase was from Megazyme (Bray, Ireland). Methyl red was from Ferak (Berlin, Germany). Polyvinylpolypyrrolidone (PVPP), ninhydrin, pectin, lipase, tannase, tannic acid, Folin–Ciocalteu reagent, sodium carbonate, dinitrosalicylic acid, bovine serum albumin (BSA) were from Sigma Aldrich (St. Louis, MO, USA). The DIAION^® HP-20 macroporous resin was from Mitsubishi Chemical (Tokyo, Japan). All chemicals were of reagent grade.

Wang S.T., Feng Y.J., Lai Y.J, & Su N.W. (2019). Complex Tannins Isolated from Jelly Fig Achenes Affect Pectin Gelation through Non-Specific Inhibitory Effect on Pectin Methylesterase. Molecules, 24(8), 1601.

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Cell Fixation and Immunostaining Protocol

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Cells were harvested with 0.5% Trypsin solution (Thermo Fischer), washed with PBS after centrifugation at 500 g for 5 min, and then resuspended in 70% ethanol solution precooled to −20°C. After an incubation at −20°C for at least 2 h, cells were centrifuged, washed 2× with PBS supplemented with 0.05% Triton X‐100 (Sigma), and resuspended in blocking buffer (PBS, 0.25% Triton X‐100, 5% FCS) for 30 min. Cells were incubated for 1 h in blocking buffer supplemented with the indicated antibody, washed 2× with washing buffer (PBS, 0.1% Triton X‐100, 5% FCS), and incubated for 1 h in blocking buffer supplemented with the appropriate secondary anti‐IgG antibody, conjugated to Alexa 488. Following 2× washes with washing buffer, cells were resuspended in PBS supplemented with 50 μg/ml propidium iodide (PI; Sigma) and 20 μg/ml RNAse A (Thermo Fischer). Flow cytometry was carried out in a FACSCalibur instrument (BD Biosciences). If no antibody staining was performed, blocking and antibody incubation steps were omitted.

Stier A., Gilberto S., Mohamed W.I., Royall L.N., Helenius J., Mikicic I., Sajic T., Beli P., Müller D.J., Jessberger S, & Peter M. (2023). The CUL4B‐based E3 ubiquitin ligase regulates mitosis and brain development by recruiting phospho‐specific DCAFs. The EMBO Journal, 42(17), e112847.

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Synthesis and Characterization of Amphiphilic Copolymers

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Hydrophobic Dorzolamide and IMC were purchased from Sigma Aldrich (St. Louis, MO, USA). All solvents were used as received without further purification. The human dermal fibroblast cell line (HDFa) and the necessary supplies (antibiotic cocktail: penicillin and streptomycin; non-essential amino acids; trypsin solution; and foetal bovine serum—FBS) for the in vitro cytotoxicity assay were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Two copolymer samples were investigated in the present study. The PCL-g-P(NVCL-co-NVP) copolymers were obtained by a grafting-from technique in three steps, as previously reported [24 (link)]. The backbone was synthesised by ring-opening copolymerisation of ε-caprolactone (ε-CL) and α-chloro-ε-caprolactone (α-Cl-ε-CL). In the second step, a RAFT macroinitiator was prepared by the substitution of pendant chloro groups with a xanthate salt. Finally, RAFT-Madix copolymerisation of N-vinyl caprolactam (NVCL) with N-vinylpyrrolidone (NVP) was carried out. The obtained copolymers were analysed by ¹H NMR (Bruker AC-400F, MA, USA) and size exclusion chromatography (Shimadzu LC-20AD liquid chromatograph, Japan), and the molecular characteristics of the two samples are provided in Table 4.

Ozturk M.R., Popa M., Rata D.M., Cadinoiu A.N., Parfait F., Delaite C., Atanase L.I., Solcan C, & Daraba O.M. (2022). Drug-Loaded Polymeric Micelles Based on Smart Biocompatible Graft Copolymers with Potential Applications for the Treatment of Glaucoma. International Journal of Molecular Sciences, 23(16), 9382.

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Biofunctionalized Silica Nanoparticle Synthesis

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Cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS, 99.98%), (3-aminopropyl) triethoxysilane (APTES), and eugenol (4-allyl-2-methoxyphenol) were purchased from Sigma-Aldrich. Casein, trypsin solution, absolute ethanol (EtOH), succinic anhydride, 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), N,N-dimethylformamide (DMF), 2-(N-morpholino)ethanesulfonic acid (MES) buffer, and glacial acetic acid were purchased from ThermoFisher Scientific, USA. Chemical reagents were of analytical grade and were used as received.

Wijewantha N., Sane S., Eikanger M., Antony R.M., Potts R.A., Lang L., Rezvani K, & Sereda G. (2023). Enhancing Anti-Tumorigenic Efficacy of Eugenol in Human Colon Cancer Cells Using Enzyme-Responsive Nanoparticles. Cancers, 15(4), 1145.

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Cell Culture, Migration, and Proliferation Assays

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The established cell cultures were cultivated until the confluency grade became 80%; then, the cells were removed from the flask surfaces by using 0.25% trypsin solution (Thermo Fisher, Waltham, MA, USA) and reseeded on new flasks. As a cell culture media was used high-glucose DMEM with 1mM Sodium Pyruvate and 300 mg/L L-glutamine (Thermo Fisher, Waltham, MA, USA), supplied with 10% Fetal Bovine serum (FBS) (Thermo Fisher, Waltham, MA, USA), penicillin and streptomycin (Thermo Fisher, Waltham, MA, USA).
The migration and proliferation assays were considered by using a time-lapse Cell IQ machine, which is described by Narkilahti et al. [62 (link)]. This system acts as a hardware phase-contrast microscope and CO₂ incubator assembly and a software complex designed for image recognition for the quantification of proliferation and migration image data.

Shmelev M.E., Farniev V.M., Shved N.A, & Kumeiko V.V. (2023). Nanomechanical Signatures in Glioma Cells Depend on CD44 Distribution in IDH1 Wild-Type but Not in IDH1R132H Mutant Early-Passage Cultures. International Journal of Molecular Sciences, 24(4), 4056.

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