0.22 m pvdf filter

Twenty-five kilograms of fresh tea leaves of the Camellia sinensis (Jin Guanyin) variety (one bud with two or three leaves) were collected from the tea garden at Qilin Mountain Tea Factory, Fujian, China. These tea leaves were then divided into five portions for the manufacturing of DL, GT, OT, BT and WT at the tea factory of Fujian Agriculture and Forestry University by the manufacturing procedures illustrated in Figure S1.
For cell culture experimentation, 25 g of dried tea powder were extracted with water for 30 min at 80 °C and then freeze-dried into powder after filtering. The tea extracts were stored at −80 °C and diluted by PBS until use.
Chemical profiling analysis was performed according to previous methods [49 (link),50 (link)]. Briefly, freeze-dried tea leaves were individually ground to fine powders using precooled mortar and pestle. Following lyophilization, 30 mg (±0.5 mg) of ground samples were weighed and 1.2 mL of 70% (v/v) methanol was added for metabolite extraction. Samples were vortexed, sonicated at 25 °C for 20 min and centrifuged (10 min, 12,000 g). Supernatants were diluted 50-fold with 70% (v/v) methanol, filtered through a 0.22 µm PVDF filter (Millipore) and stored at −20 °C until analyzed. Three biological sample replicates were prepared for each tea types.

EVs were collected by density gradient ultracentrifugation as described previously.^[62] THP‐M culture medium was centrifuged at 800 × g for 5 min, followed by centrifugation at 2000 × g for 10 min to eliminate cellular debris. The supernatant was then passed through a 0.22‐µm PVDF filter (Millipore, USA) and centrifuged at 100 000 × g for 90 min at 4°C. The supernatant was discarded, and the pellets were washed with PBS and used to suspend the pellets. EVs were isolated from plasma of patients with NSCLC by use of Minute Hi‐Efficiency Exosome Precipitation Reagent (Invent Biotechnologies, USA). For removing large debris, 1‐mL plasma samples were centrifuged for 10 min at 2000 × g. Two volumes of EVs precipitation reagent were added to the supernatant, followed by 1 h of incubation. Purified EVs were obtained by centrifugation of the sample for 30 min at 10 000 × g, followed by centrifugation at 100 000 × g for 70 min. The size distribution and concentration of EVs were determined with a ZetaView particle tracker from ParticleMetrix (Germany). The shape of EVs was assessed with a transmission electron microscope (Tokyo, Japan). CD63, Alix, and TSG101 served as markers of EVs; GM130 served as a marker of cells. The concentrations of EVs were quantified with BCA protein assay kit (Beyotime Biotechnology, China).

R&D system Luminex Kit was used to analyze cytokine's secretion both in sera obtained from BM aspirates of young and aged donors and in conditioned media (CM) derived from young and aged MSC. Sera from 6 BM of young adults and 8 BM of aged subjects were collected from BM aspirates and analyzed prior MSC derivation. For CM collection, MSC from young and aged donors were plated the day before at 4 × 10⁵ cells/well in a 6‐well plate in StemSpan (Stem Cell Technologies) without addition of other factors for 48 hr. Media were then collected, filtered with a 0.22µm PVDF filter (Millipore), and stored at −80°C to use for Luminex or CM experiments.
Customized Luminex plates were obtained to screen for: IL1α, IL8, IL6, MCP1, IL1β, TNF‐α, GROβ, and CCL4. Assays were run as per manufacturers’ instructions with standards and samples in technical duplicates. Data were acquired on a calibrated Bio‐Plex MAGPIX multiple reader system (Bio‐Rad) and visualized with Bio‐Plex manager Software.

Oral bacteria, including Actinomyces naeslundii NC-3, Streptococcus gordonii DL1, S. cristatus CC5A, S. mutans KPSK2, Actinomyces viscosus EG4, and Porphyromonas gingivalis 33277, which were from our laboratory collection at Meharry Medical College School of Dentistry, were grown in appropriate media aerobically or anaerobically at 37°C for 16 h. Bacterial cells were harvested by centrifugation at 6,000×g for 10 min at 4°C. Bacterial cells were washed twice with cold PBS. The cell extracts were then prepared by sonication at Power 15 (10×30 sec) using a Misonix XL-2000. The supernatants were collected and filtered through a 0.22 µm PVDF filter (Millipore). The bacterial extracts were stored in −20°C freezers if long term storage were needed.

Samples (100 mg) were homogenized in 1 mL of ethanol/deionized water (50:50, v/v) and extracted at 70 °C for 20 min following the procedure described previously. All the obtained extracts were centrifuged at 1520× g for 5 min, filtered through a 0.22 µm PVDF filter (Millipore, MA, USA), and kept at −20 °C until posterior chromatographic analysis.

Culture medium was centrifuged at 3000g for 15 min, and filtered through a 0.22-µm PVDF filter (Millipore) to remove cells and cellular debris. Then the filtered culture medium was mixed with the Exoquick exosome precipitation solution (System Biosciences, CA, USA) at a ratio of 1:5 and refrigeration for at least 12 h. Thereafter, the mixture was re-centrifuged at 1500g for 30 min, the supernatant was discarded and exosomes were collected. Purified exosomes were resuspended in approximately 100 μL of PBS and subjected to transmission electron microscopy (TEM) (× 200) (JEOL, Akishima, Japan), cell co-culture, RNA extraction with Trizol reagent or protein detection with RIPA lysis buffer [30 (link)].