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2 protocols using immobilized tpck trypsin

1

Caco-2 Cell Culture and Supernatant Preparation

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The Caco-2 cells were routinely grown at 37°C in a 95% air–5% CO2 atmosphere in Dulbecco's modified Eagle medium (DMEM) with high glucose (Sigma-Aldrich Co) and 10% heat-inactivated (56°C, 30 min) fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). The cells were harvested using trypsin-EDTA (Gibco) and suspended with DMEM-10% FBS to stop the reaction with trypsin-EDTA. The cells were collected from the cell suspension by a centrifugation, and were washed with Dulbecco's phosphate buffered saline (DPBS; Gibco). Then, the cells were resuspended with DMEM without any serum, and the mixture was centrifuged twice. Finally, the cellular suspension in DMEM was adjusted to 1.0 × 105 cells per 75 cm2 of the tissue culture flask (Thermo Scientific, Waltham, MA, USA). The culture medium was replaced every 5 days, and the supernatant collected after 5, 10, and 15 days of culture was used in subsequent experiments. The collected supernatant was filtered with Centriprep YM-3 or YM-10 (3 or 10 kDa; Millipore, Bedford, MA, USA). For some experiments, 500 μl of the various Caco-2 cell supernatant samples were treated with 25 μl of Immobilized TPCK Trypsin (Thermo Fisher) for 1 h at 37°C; the Immobilized TPCK Trypsin was then removed by centrifugation (10,000 × g for 10 min).
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2

Synthesis and Characterization of CPPM Compound

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Dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific and DMSO-d6 was purchased from Cambridge Isotopes Laboratory, Inc. PBS Buffer, 10x, pH 7.4, was purchased from Thermo Fisher Scientific, Inc. and diluted to 1x concentration with Milli-Q water. Trifluoroacetic acid (TFA) was purchased from Alfa Aesar and used without further purification. Methanol and dichloromethane were purchased from Fisher Scientific and used without further purification. Additional materials required for the synthesis of the CPPM used in this study, MePh10-b-dG5, have been previously reported.43 (link),44 (link) Human angiotensin II (#A9525), bradykinin (#B3259), diethylpyrocarbonate (DEPC) (#D5758), imidazole (#I5513), sodium chloride (#S5886), guanidine thiocyanate (GTC, #G6639), and iodoacetamide (#I6125) were all purchased from Sigma-Aldrich (St. Louis, MO). Immobilized TPCK-trypsin (#20230) was obtained from Thermo Scientific (Waltham, MA). Potassium phosphate monobasic (#P285), sodium phosphate dibasic anhydrous (#S374), formic acid (#A117), water (#W7), and acetonitrile (#A998) were obtained from Fisher Scientific (Fair Lawn, NJ). Amicon® centrifugal filters (#UFC501096 & #UFC800324) were from Millipore Sigma (Burlington, MA). Superfolder green fluorescent protein (sfGFP) was expressed and purified by Dr. Francesca Anson using procedure described below.
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