Durapore pvdf membrane

Six million labeled cells were seeded in 55-cm² cell culture dishes and maintained overnight for cell attachment in the RPMI1640 medium supplemented with 10% fetal bovine serum, 20 mM L-glutamine (either ¹⁴N or amide-¹⁵N-Gln). After 12 h, the culture medium was removed, and the cells were washed for 3 times with phosphate-buffered saline. And then 10 mL of serum-free, phenol red-free RPMI1640 medium with 20 mM L-glutamine (either ¹⁴N or amide-¹⁵N) was added to each dish. After incubation for 48 h, the conditioned medium was collected and filtered through a 0.22 µm Durapore PVDF Membrane (Millipore, Billerica, MA, USA). Before, during and after serum starvation, viability of cancer cells was assessed using the trypan blue dye exclusion assay. The protein concentrations of conditioned media were determined using a BCA protein assay kit (Pierce,Rockford, IL). The conditioned medium with equal protein amount obtained from SKOV3 and SKOV3-ip were mixed. Subsequently, the mixtures were concentrated by Amicon Ultra-15, 3,000 molecular weight cut off (MWCO) centrifugal filter units (Millipore, Billerica, MA, USA) with excessive salt removing for quantitative glycomics. All samples were stored at −80°C until further use.

Fresh lotus seeds were dried using intermittent hot air drying with 2.0 m/s at 65 °C for 72 h. The lotus plumule was separated and milled into a powder form. The dried powder was pre-treated with anhydrous ethanol at 50 °C to remove pigments and other small alcohol soluble molecules. The pre-treated powder (200 g) was mixed with distilled water (1:10, w/v) at 92 °C for 5 h. The extract (supernatant) was collected by centrifugation at 900× g for 15 min. The remaining powder was mixed again with distilled water at 92 °C for 5 h and the extract was collected by centrifugation. Supernatants from both extraction steps were combined, concentrated under reduced pressure at 55 °C, and precipitated with three volumes of ethanol (95%, v/v) for 24 h at 4 °C. The precipitate was separated by centrifugation (900× g, 15 min) and deproteinization by employing the Sevag reagent (chloroform/n-butanol 4:1, v/v) to obtain NNP. After filtration using a Durapore^® PVDF membrane (0.45-μm, Millipore, Bedford, MA, USA), NNP were fractionated using gel permeation chromatography with a DEAE-cellulose DE-52 column (2.6 cm × 40 cm) and eluted with 0, and 0.5 mol/L NaCl solutions at a flow rate of 1 mL/min. The fractions were marked as NNP-1 and NNP-2 and assayed using the phenol–sulfuric acid method. The main fraction NNP-2 was collected, lyophilized, and used for further characterization.

A paper cigarette (Peace; Japan Tobacco Inc., Tokyo, Japan; tar, 28 mg; nicotine, 2.3 mg) with the filter removed was ignited and sucked using a peristaltic pump; the tobacco smoke produced was bubbled into cell culture medium (i.e., DMEM). Each cigarette was smoked in 6 min, and 10 ml of cell culture solution was used for each cigarette. The solution was filtered through a sterilization filter (Stericup [250 ml]; Durapore PVDF membrane [0.45 μm]; Millipore, Burlington, MA, United States) and used as the CSE. For cell death experiments, 25% CSE was used, while 5% CSE was used to confirm whether a change in mitochondrial function occurred at a concentration at which cell death did not occur.

HEK293T cells were grown on 150-mm dishes (CORNING) coated with poly-l-lysine (Sigma) until 90% confluency. For each 150-mm dish, 21 μg of the library plasmids, and 15 μg of psPAX2 (Addgene 12,260) and 6 μg of pMD2.G (Addgene 12,259) were transfected into HEK293T cells using 63 μl of Neofect DNA transfection reagent (Neo Biotech). At 16 h post transfection, the culture medium was changed with viral production medium (Lonza). Virus supernatant was collected 40 h post transfection, filtered with a 0.45-μm Sterile Filter Unit with Durapore PVDF Membrane (Millipore), aliquoted, and stored at − 80 °C before use.