Millex gp 0.22 μm filter

The rice koji was dried using a freeze dryer and ground using a mortar. Each sample powder (3 g) was extracted with 30 mL of 80% aqueous methanol by sonication for 10 min, and then shaken at 200 rpm for 24 h. Next, the sample mixtures were centrifuged at 5000 rpm for 10 min at 4 °C. The supernatants were filtered using Millex^®GP 0.22 μm filters (Merck Millipore, Billerica, MA, USA) and dried in a speed vacuum concentrator (Biotron, Seoul, Korea). The extraction yield from each sample was calculated. For the GC-TOF-MS analysis, norvaline was added as an internal standard, and the sample mixture was derivatized. One hundred microliters of methoxyamine hydrochloride (20 mg/mL in pyridine) was added to each dried sample, and the samples were heated at 30 °C for 90 min. Next, 100 μL of the derivatization agent MSTFA was added to each sample, and the derivatization reactions were heated at 37 °C for 30 min. For UHPLC-LTQ-IT-MS/MS, formononetin was added as an internal standard. The dried samples were dissolved in 200 μL of 80% aqueous methanol, and filtered using 0.2 μm polytetrafluoroethylene (PTFE) filters.

For derivatization, 100 μl of the supernatant was taken in a fresh e-tube and completely dried. First, the oximation was performed by adding 50 μl of methoxyamine hydrochloride (20 mg/ml in pyridine) to the dried extract and incubated at 30°C for 90 min. Next, the silylation was performed by adding 50 μl of MSTFA to the reaction mixture, followed by a 37°C incubation for 30 min. The final concentration of the derivatized samples was set at 20,000 ppm; and daidzein (0.25 mg/ml) was used as the added internal standard (IS). All samples were filtered using Millex-GP 0.22-μm filters (Merck Millipore, Billerica, MA, USA) prior to the instrument analyses.
GC-TOF-MS analysis was performed using an Agilent 7890A GC system (Agilent Technologies, Palo Alto, CA, USA) coupled with an Agilent 7693 autosampler (Agilent Technologies) and a Pegasus HT TOF-MS (Leco Corporation, St. Joseph, MI, USA). The chromatographic separation was conducted by an Rtx-5MS column (30 m length × 0.25 mm inner diameter; J&W Scientific, USA) with a helium as carrier gas at a constant flow (1.5 ml/min). The analytical program for sample analysis was adopted from a previous study (19 (link)). We utilized three biological replicates for each type; and the analyses were performed in random order to reduce the bias and systematic errors.

For lentiviral vector production, transfection of 293T cells was performed by using the calcium phosphate method in the presence of 15 mM HEPES (PAA) and 25 μM chloroquine (Sigma-Aldrich). Lentiviral vector plasmids were transfected with expression plasmids for human immunodeficiency virus (HIV) group-specific antigens and viral enzymes (Gag/Pol; pcDNA3.GP.4xCTE), regulator of expression of virion proteins (Rev; RSV-Rev; kindly provided by Thomas Hope, Chicago, IL, USA), and the envelope protein vesicular stomatitis virus (VSVg) (all helper plasmids produced by Plasmid Factory, Bielefeld, Germany). Briefly, 5 × 10⁶ 293T cells were transfected with 5 μg of the LV vector, 12 μg of Gag/Pol, 5 μg of Rev, and 1.5 μg of VSV-G-encoding plasmids; 36–48 h after transfection, supernatants containing lentiviral particles were harvested and filtered through Millex-GP 0.22 μm filters (Millipore, Darmstadt, Germany).

Conditioned media from each of the three categories were harvested after 48 h and centrifuged for 30 min at 800 g to remove any floating and unattached cells. In order to remove the cellular debris, the supernatants were again filtered by running through a Millex-GP 0.22 μm filter (Millipore, Ireland). Following this, filtered secretomes were concentrated using a 3 kDa cut-off Millipore Amicon Ultra filters (Millipore, Billerica, MA, USA) by centrifugation at 4000 g till the secretome was condensed to 1mL, and subsequently centrifuging at 14,000 g using the 3 kDa cut-off filters till a condensed volume of 500µL was obtained. Pierce microBCA kit (Thermo Scientific) was used to determine the protein concentration.