Himac cp80wx

After an incubation at 55°C for several d when the OD₆₀₀ value was >0.15, cells were collected via centrifugation at 5,000×g at 4°C for 10‍ ‍min during the stationary phase. The cell pellet was centrifuged again after being washed with saline containing 8.5‍ ‍mg L^–1 NaCl. The pellet was suspended in 10‍ ‍mL of 10‍ ‍mM potassium phosphate buffer (KPB, pH 7.0) and subjected to 16,000 psi of pressure in a French press (American Instrument Company). Debris was removed via centrifugation at 8,000×g at 4°C for 15‍ ‍min, and the supernatant was ultracentrifuged at 100,000×g at 4°C for 60‍ ‍min using an ultracentrifuge (himac CP80WX, Hitachi). The resulting precipitate was homogenized in 10‍ ‍mL of 10‍ ‍mM KPB and used as the membrane fraction. The collected supernatant was dialyzed against 1 L of 10‍ ‍mM KPB at 4°C every 6 h. The treated solution was used as a soluble fraction.

Conditioned cell media were collected, followed by centrifugation at 800 g for 5 min and 12000 g for 20 min at 4 °C. Supernatants were filtered through a 0.22-μm pore filter (Millipore, USA) and ultracentrifuged (Himac CP-80WX, Hitachi, Japan) at 110000 g for 3 h at 4 °C. Pellets were collected, resuspended in HBSS, and ultracentrifuged at 110000 g for 2 h at 4 °C. Pellets were resuspended in HBSS or RIPA buffer and stored at -80 °C until use.

Caco-2 cells were treated with or without 2 mM IIAEK for 24 h. Following treatment, the cells were washed twice with ice-cold phosphate-buffered saline and collected in 1 mL of cold TNE buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EGTA, pH 7.5) + 1% (w/v) Triton X-100. The cell lysates were homogenized on ice using a glass homogenizer (WHEATON, 903475) with an attached potter-type shuttle (WHEATON, 358034). The homogenate was transferred to the 13PA tube (HITACHI: 332001A), and 2 mL of 80% (w/v) sucrose in TNE buffer was added. Homogenates were layered with 6 mL of sucrose density gradient solution (5–30%) using a fractionator, followed by ultracentrifugation at 200,000× g and 4 °C for 17 h in an ultracentrifuge (Himac, CP80WX, HITACHI, Tokyo, Japan) with a swing rotor (Himac, P40ST, HITACHI), as previously described [17 (link)]. Subsequently, samples were fractionated into 10 fractions and recovered using the density gradient fractionator MODEL DGF-U (HITACHI). Sucrose density gradient formation was confirmed by measuring the sucrose density (w/w %) of each fraction with the ATAGO pocket sugar meter (ATAGO). Each fraction was subjected to ultrafiltration using an Amicon^® Ultra 10K device (Millipore) according to the manufacturer’s protocol. The obtained concentrated protein solutions obtained were used for photoaffinity labeling.

Exosomes were purified by differential centrifugation procedures, as described previously [13 (link), 41 (link)]. Supernatants were collected from cells that had been cultured for 48 h in medium containing exosome-depleted FBS, and they were subsequently subjected to sequential centrifugation steps at 300g for 10 min, 2,000g for 10 min and 10,000g for 30 min to remove cell debris, dead cells and EVs other than exosomes. Supernatants were then centrifuged at 100,000g for 70 min at 4°C (himac CP80WX, Hitachi, Ltd., Tokyo, Japan). The pelleted exosomes were suspended in PBS and collected by ultracentrifugation at 100,000g for 70 min. The purified exosomes were resuspended in PBS and used in subsequent experiments.
Exosome abundance was estimated with the ExoELISA-ULTRA CD63 kit (System Bioscience, LLC.) according to the manufacturer’s protocol. This assay is a sensitive, direct Enzyme-Linked ImmunoSorbent Assay (ELISA) to quantitate exosome abundance in a given sample. The amount of exosomes is estimated by detecting CD63 on the exosome surface by specific antibody.