Amicon ultra centrifugal filter devices 100k

The bacterial inoculum (500 µL) of S. aureus C-29 was inoculated into 250 mL of BHI broth and incubated at 37 °C for 6, 17, and 24 h with shaking (110 rpm). The culture solution obtained at each time was transferred to 50 mL polypropylene sterile disposable centrifuge tubes and centrifuged (3000× g, 15 min). After the bacterial cell pellet was removed, the supernatant was filtered through a 0.2 µm filter (Advantec Toyo Co., Ltd., Tokyo, Japan). The filtrate was transferred to Amicon Ultra centrifugal filter devices 100K (Merck, Darmstadt, Germany) and centrifuged (4000× g, 30 min). A 100 kDa cutoff (>100 kDa) concentrate was added to the ultracentrifugation tubes (11PA thick-walled tubes, Hitachi Koki Co., Ltd., Tokyo, Japan) and ultracentrifuged (150,000× g, 4 °C, 3 h) using the Himac CP-WX Series separation ultracentrifuge (Hitachi Koki Co., Ltd., Tokyo, Japan). The pellet was resuspended in 100 µL of PBS, and the amount of protein was measured using a micro-spectrophotometer (Nikko-Hansen Co., Ltd., Osaka, Japan). The isolated MVs were stored at −20 °C until use. Unless otherwise stated, the MVs were used as undiluted.

A bacterial suspension (500 µL) was introduced into brain heart infusion (BHI) broth containing a 0.3 mM polyphenol solution (250 mL) and incubated at 37 °C for 17 h with shaking at 110 rpm. After incubation, the resulting culture solution underwent centrifugation (11,600× g, 5 min). The supernatant was then filtered through a 0.2 µm filter from Advantec Toyo Co., Ltd., Tokyo, Japan. The filtered culture supernatant was further processed by fractionation at 100 kDa using a centrifugal ultrafiltration filter unit (Amicon Ultra centrifugal filter devices 100 K; Merck Millipore, Darmstadt, Germany) employing the Himac CP-WX Series separation ultracentrifuge (Hitachi Koki Co., Ltd., Tokyo, Japan). The >100 kDa concentrate obtained was subjected to additional ultracentrifugation (150,000× g, 3 h) to isolate microvesicles (MVs) in the sediment fraction. The protein concentration of the isolated MVs was determined using a K2800 micro-spectrophotometer from Beijing Kaiao Technology Development Co., Ltd. The isolated MVs were stored at −20 °C until use, and unless specified otherwise, they were utilized in their undiluted form.