The selected protein spots were excised, mixed with 20 ng/mL trypsin (Amersham, Picataway, NJ, USA), incubated at 37 °C for 16 h, and extracted with 1% trifluoroacetate. The peptide footprint of the extract was characterized using Ultraflex™ matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (Bruker Daltonics, Hamburg, Germany). Match peptide mass were performed using MASCO bioinformatics. The criteria of candidate protein were that the peptide mass showed MASCOT score ≥ 65, along with a sequence coverage of ≥ 20% of the matched peptides of the Swiss-Port database. The MOWSE score ≥ 39 of the match peptide was considered as homology of the match protein [48 (link)].
Trypsin
Manufactured by Cytiva
Sourced in United States
Trypsin is a proteolytic enzyme commonly used in cell culture applications to detach adherent cells from the cell culture vessel. It works by cleaving the peptide bonds of specific amino acid residues, allowing cells to be separated and dispersed for further processing or subculturing.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Cytiva (14 mentions)
Dulbecco s modified eagle s medium dmem, by Cytiva (9 mentions)
Penicillin streptomycin, by Cytiva (6 mentions)
Nf κb p65, by Cell Signaling Technology (4 mentions)
Streptomycin, by Cytiva (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Cytiva (3 mentions)
Cleaved caspase 9, by Cell Signaling Technology (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Nf κb p50, by Santa Cruz Biotechnology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ho 3867, by MedChemExpress (2 mentions)
Rpmi 1640 medium, by Cytiva (2 mentions)
Penicillin, by Cytiva (2 mentions)
Potassium cyanide kcn, by Merck Group (2 mentions)
Cytotoxicity detection kit ldh, by Roche (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
45 protocols using trypsin
1
Protein Identification via MALDI-TOF MS
2
Isolation and Survival Assay of Neonatal Rat Ventricular Myocytes
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Neonatal rat ventricular myocytes (NRVMs) were freshly isolated using an established protocol68 (link). Left ventricles collected from 2-day-old Sprague–Dawley rats (Harlan) were digested by trypsin (Amersham Biosciences) and collagenase type II (Worthington Biochemical). Isolated cells were re-suspended in the M-199 medium (Life Technologies) supplemented with 10% FBS, 19.4 mM glucose, 2 mM l-glutamine, 2 U/mL penicillin, 0.8 μg/mL vitamin B12, 10 mM HEPES, and 1 × MEM nonessential amino acids (Sigma-Aldrich). Two rounds of 60 min pre-plating were performed, during which cardiac fibroblasts attach to the dish bottom, thus enriching the nonadherent population for NRVMs. The nonadherent cells (NRVMs) were then seeded onto the different collagen matrices in 24-well plates (40,000 cells/cm2). For the survival assay, 3-day cultured NRVMs were subjected to M-199 media containing 50 µM hydrogen peroxide for 3 h, after which a Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay was performed and dead cells were counted in three random fields-of-view.
3
RBL Cell Culture and Signaling
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RBL cells (rat basophilic leukemia) were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA). Sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO), DNP IgE, DNP-HSA, calcium ionophore A23187, PMA, compound 48/80, 4-nitrophenyl-N-acetyl-β-D-glucosaminide, lipoxygenase from Glycine max (soybean), linoleic acid, (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), Evans blue, toluidine blue O, DNCB, and formaldehyde solution were bought from Sigma (St. Louis, MO, USA). Trypsin (0.25%) was purchased from HyClone Laboratories (Logan, UT, USA). Additionally, TRI Reagent® solution was acquired from Molecular Research Center, Inc. (Cincinnati, OH, USA). 1X phosphate-buffered saline (PBS) was purchased from Samchun Pure Chemical Co. (Gyeonggi-do, Korea). The sets of primers for quantitative real-time polymerase chain reaction (PCR) were synthetized by Macrogen (Seoul, Korea). Horse anti-mouse HRP-conjugated secondary antibody, goat anti-rabbit HRP-conjugated secondary antibody, and the antibodies against the total and phosphorylated forms of Syk, PLCγ1, PKCδ, PI3K, AKT, NF-κB p65, p38, JNK, and ERK1/2 were acquired from Cell Signaling Technology (Beverly, MA, USA), and those against NF-κB p50 and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
4
Developing Adriamycin-Resistant Breast Cancer Cells
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Human breast epithelial cell line MCF-10A, adriamycin (ADR)-sensitive BC cell line MCF-7 (MCF-7/S), and HEK293T cells were all procured from American Type Culture Collection (Manassas, VA). Subsequently, MCF-7 cells were exposed to ADR of increasing concentrations in order to develop BC cell line MCF-7 resistant to 500 nM ADR (MCF-7/ADR). These cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin in a 5% CO2 incubator (BB15, Thermo Fisher Scientific Inc., Waltham, MA) at 37°C. Next, the medium for MCF-7/ADR was supplemented with 1.0 μmol/L of ADR, with the culture medium refreshed every 24 h. Afterwards, the cells were detached with 0.25% trypsin (HyClone Laboratories, Logan, Utah) every 72 h, passaged and collected.
5
Evaluating Anticancer Activity of HF-TPGS Nanoparticles
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HF was purchased from Shanxi Meixilin Pharmaceutical Co., Ltd. (Yuncheng, China). TPGS was purchased from Guangzhou Kafen Biological Technology Co., Ltd., (Guangzhou, China). PTX was purchased from Dalian Meilun Biological Technology Co., Ltd. (Dalian, China). Fetal bovine serum (FBS) and an MMP detection kit were provided from Nanjing Shanben Biotechnology Co., Ltd., (Nanjing, China). Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin, trypsin, and streptomycin were obtained from Hyclone Laboratories (Logan, UT, USA). An apoptosis detection kit, cell cycle detection kit and ROS detection kit were purchased from Nanjing Kaiji Biotechnology Co., Ltd (Nanjing, China). All other reagents and chemicals were of analytical grade.
6
Cell Differentiation Induction Protocol
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Tris, glycine, NaCl, SDS, mannitol, bovine serum albumin (BSA), sodium hydrosulfide (NaHS), β-glycerophosphate (β-GP), ascorbate, and propargylglycine (PAG) were purchased from Sigma-Aldrich (St Louis, MO, USA). HO-3867 and LY3000328 (LY) were from Medchem express, LLC (Princeton, NJ, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovineserum (FBS), streptomycin/penicillin and trypsin were obtained from Hyclone Laboratories (South Logan, UT, USA). The RIPA buffer was purchased from Thermo Fisher Scientific Inc (Waltham, MA, USA). The Bradford colorimetric protein assay kit (Rockford, IL, USA) was used for protein quantification. The calcium, phosphorus, and alkaline phosphatase (ALP) kits were purchased from Jiancheng Bioengineering Co (Nanjing, China).
7
Nanoformulation for Cancer Treatment
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Shanxi Meixilin Pharmaceutical Co., Ltd. (Yuncheng, China) provided the halofuginone hydrobromide (HF). We purchased D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) from Guangzhou Kafen Biological Technology Co., Ltd. (Guangzhou, China) and poloxamers 188 and 407 from Beiersdorf Biological Technology Co., Ltd. (Hamburg, Germany). Gibco Laboratories (Grand Island, NY, USA) provided fetal bovine serum (FBS). We obtained Roswell Park Memorial Institute (RPMI) 1640 medium, penicillin, trypsin, and streptomycin from Hyclone Laboratories (Logan, UT, USA). We purchased Ki-67, STC-1, VEGF, CD31, BRCA2, TGF-β，Collagen I and Vimentin antibodies and an immunohistochemistry kit from Abcam Biotechnology Co., Ltd. (London, England). We used other chemicals and reagents of analytical grade without further purification.
8
TPA-Induced Cell Proliferation Assay
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Cells were plated in 24-well cell culture dishes in DMEM. After 24 hours, the medium was changed to DMEM supplemented with 100 nM TPA (Sigma-Aldrich Canada Co.) or vehicle control (DMSO). The medium was then replaced every 2 days for 6 days. On day 6, cells were detached from three separate wells using 0.05% trypsin (Hyclone Laboratories Inc.) and counted using the TC-1 counter (Bio-Rad Laboratories Inc.).
9
Fabrication and Characterization of Heparin-Coated Stainless Steel Substrates
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316L stainless steel (SS) substrates were processed by Chengdu Derbo Steel Co., Ltd; dopamine hydrochloride, pAM solution, DOTA, and copper (II) chloride hydrate (CuCl2·2H2O, 99%) were purchased from Aladdin Reagents Co., Ltd.; ethanol, isoamyl acetate, hydrochloric acid, acetone, sodium chloride and sodium hydroxide were purchased from Chengdu Kelong Chemical Reagents Co., Ltd.; quartz sensor crystals were purchased from Jiaxing Jingkong Electronic Co., Ltd.; N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 2-(N-morpholino) ethanesulfonic acid hydrate (MES) were purchased from Sigma-Aldrich Corp.; NHS-Fluorescein isothiocyanate (NHS-FITC, 90%) was bought from Thermo Fisher Technology Co., Ltd.; phosphate buffered saline solution (PBS), Tris-buffered saline solution (TBS) and trypsin were purchased from Hyclone Laboratories; Cell culture medium for ECs and SMCs were purchased from Vasculife® by Lifeline Cell Technology Co., Ltd.; Chromogenix Coamatic® Heparin test, used to measure the anticoagulant activity of heparin, was purchased from Chromogenix Co., Ltd.; activated partial thromboplastin time (APTT) reagent was purchased from Shanghai Sun Biotech Co., Ltd.; mouse anti-human fibrinogen/HRP, goat anti-mouse IgG/HRP, and anti-fibrinogen gamma chain antibody were purchased from Beijing Biosynthesis Biotechnology Co., Ltd.
10
Cytotoxicity and Proliferation Assay
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Crystal violet (N-hexamethylpararosaniline), hexadecyltrimethylammonium bromide (CTAB) and albumin were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). Trypsin, and penicillin/streptomycin were obtained from HyClone Laboratories (Utah, USA). The Cytotoxicity Detection Kit (LDH) and the Cell Proliferation ELISA, BrdU (colorimetric) Kit were obtained from Roche Applied Science (Germany). Potassium cyanide (KCN) was obtained from Merck (Darmstadt, Germany). All the other chemicals were of reagent grade and purchased from common commercial suppliers.
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