Polyvinylidene fluoride filter

The plasma concentration of CoQ10 was determined as described in our previous study.^{[17 (link),18 (link)]} Briefly, frozen plasma samples were thawed on ice and then maintained at 4°C during handling. Thawed plasma was pipetted into Eppendorf microcentrifuge tubes, deproteinized with methanol, and then treated with n-hexane. The mixture was vortexed for 5 minutes and then centrifuged at 2500 × g and 4°C for 15 minutes, after which the clear n-hexane layer was transferred to another tube for another round of n-hexane extraction. The plasma extracts were combined and evaporated to dryness under a gentle stream of nitrogen gas. The dry residue was dissolved in the mobile phase, filtered through a membrane (polyvinylidene fluoride filter, 4 mm, 0.45 μm; Millipore, Bedford, MA), and injected into a high-performance liquid chromatography system. A microBondapak C18 3.9-mm × 30.0-cm stainless steel column with a 3 × 22–mm guard column packed with microBondapak C18 was used with the mobile phase of methanol–n-hexane 85:15 (v/v). The flow rate was 1 mL/min. The wavelength of the ultraviolet detector was fixed at 276 nm. To determine the amount of CoQ10 in plasma, a calibration curve was constructed by plotting the peak areas versus the concentration. Linearity was achieved in the concentration range of 0.12 to 1.92 μg/mL.

BPN‐BBTD was synthesized according to the literature, with minor modifications.^[30] The different micelles were prepared using a nanoprecipitation method, and the amounts of ingredients are shown in Table S4 (Supporting Information). In a typical procedure for the NIR‐II CL nanoprobe, different amounts of F127, CPPO, PFODBT, and BPN‐BBTD were dissolved in THF (1 mL) to obtain a stock solution. Under intense sonication for 2 min, the stock solution was quickly injected into the water to produce a clear micellar solution. After THF was removed by heating the micelle solution at 45 °C for 12 h, to get rid of big aggregates, the aqueous solution was filtered through a polyvinylidene fluoride filter (0.22 µm, Millipore, USA). The obtained suspension was finally concentrated to 500 µL through ultrafiltration (Amicon Ultra‐15 30 KD, Millipore, USA) and used immediately for experiments.

Serum and cell medium were treated with polyvinylidene fluoride filter (Millipore, Billerica, Mass). Then subsequently ExoQuick solution (System Biosciences, Mountain View, CA) was added incubating at room temperature for 0.5 h. By centrifugation at 1500g for 30 min. Exosome pellets were collected and resuspended in 25 μl DEPC water.

For preparation of lentiviral particles, HEK293T cells were seeded at 50% confluency in a T75 flask one day before transfection. 4 μg of plasmid pUltra-Chili-ITGA2-HA (or ITGA2-mutants) and 2 μg of pMD2.G (Addgene #12259) and 2 μg of pCMVR8.74 (Addgene #22036) were co-transfected using 24 μL of jetPEI reagent in 1 mL of 150 mM NaCl solution (Polyplus-transfection, Chemie Brunschwig AG, Switzerland). Medium was changed 24 h after transfection. Virus supernatant was collected 48 h later and filtered with a 0.45 μm polyvinylidene fluoride filter (Millipore). 3 mL of lentivirus-containing medium was used to transduce previously established ΔITGA2 ovarian cancer cells (Huang et al., 2020 (link)) to preclude the functional impact of endogenous ITGA2 expression. dTomato + transduced cells were sorted by BD FACS Aria Cell Sorter (BD Bioscience). In addition, pUltra-Chili-ITGA2-HA and pUltra-Chili-10NQ-HA plasmids were used to transduce OVCAR4 cancer cell lines. Plasmid-encoding dTomato + transduced cells were sorted by BD FACS Aria Cell Sorter (BD Bioscience).