0.2 μm syringe filter

Marker analytes were accurately weighed and dissolved in methanol or methanol-DMSO (1:1) to a concentration of about 1.0 mg/mL and used as a standard solution. Each prepared standard stock solution was degassed in a sonicator and filtered through a 0.2 μm syringe filter (Pall Life Sciences, Ann Arbor, MI, USA). All stock solutions were stored in a refrigerator until the HPLC or LC–MS/MS analysis.
A sample solution for simultaneous analysis for quality control of P. pubescens leaves was prepared by dissolving 80% ethanol extract of P. pubescens leaves in 70% methanol at a concentration of 10 mg/mL. The solution was prepared by ultrasonic extraction for 60 min and then filtered through a 0.2 μm syringe filter (Pall Life Sciences, Ann Arbor, MI, USA). For the LC–MS/MS analysis, the prepared sample solution was diluted 10-fold prior to use.

The PDLSCs were cultured in 75 cm
²cell culture flasks to 80 to 90% confluence. They were washed twice with 10 mL of phosphate buffer saline (PBS) and refreshed with 10 mL of serum-free DMEM. Culture supernatant was collected after 48 hours of incubation and then centrifuged (1000 g, 5 min at 4°C) and filtered through a 0.2 μm syringe filter (Pall corporation, Port Washington, New York, US) to remove cell debris. The CM was concentrated using ultrafiltration with a cutoff of 10 kDa (Invitrogen, Carlsbad, California, US) at 5000 g for 40 minutes and stored at −80°C until used.

The aerial parts of I. suffruticosa Mill. were collected from the Siaying District, Tainan, Taiwan, in July 2015. The plant material was taxonomically identified by Professor Hsun-Shou Chang of the School of Pharmacy, Kaohsiung Medical University. A voucher specimen (Chen 5673) was deposited in the herbarium of the College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan. The aerial parts of I. suffruticosa were extracted with methanol:water (1:1) for three days at room temperature. The methanol-water extracted solution was collected and concentrated using a rotary evaporator under vacuum. Residual water and solvent were removed in a vacuum oven, after which the extract was stored at 4 °C. The I. suffruticosa aerial parts extract (ISAE) used in this study was prepared by dissolving 100 mg of the crude methanol-water extract in 1 mL of distilled water with 30 min of sonication, followed by the removal of the insoluble pellets by centrifugation. ISAE was sterilized by filtration with 0.2 μm syringe filter (Pall Corporation, USA) and then used for cell-based assays. To determine the concentration of ISAE, 1 mL of ISAE was dried under vacuum in a rotary evaporator and the weight of the resultant powder was measured to calculate the concentration of ISAE. The concentration of the ISAE stock solution was 29.2 mg/mL.