0.2 μm syringe filter

Py-LN(-apoE3) vesicles devoid of apoE3 were generated as previously described.^{25 (link)} Chloroform solutions of porphyrin-lipid, DMPC, cholesterol and distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyetheneglycol) (PEG2000-DSPE) were combined in a 10 : 45 : 40 : 5 molar ratio in a glass vial. The chloroform was dried under nitrogen and vacuum to form lipid films, which were then hydrated with PBS. The hydrated films were subjected to 9 freeze/thaw cycles prior to extrusion via a LIPEX Thermobarrel Extruder through a polycarbonate membrane (100 nm pore size) 10 times at 65 °C to yield py-LN(-apoE3). The particles were sterile filtered through a Corning® 0.2 μm syringe filters, shielded from light and stored at 4 °C prior to use.

When MDA-MB-231, SUM149, and SUM159 cells were confluent in T175 tissue culture flasks, the normal cancer cell growth media was replaced with 8 ml serum-free media (SFM) containing with 2% serum. After 24 h incubation in a tissue culture incubator, the supernatant was centrifuged and filtered through 0.2 μm syringe filters (Corning, Corning, NY). The resulting tumor-conditioned media (TCM) was stored in aliquots at −80°C. When fibroblasts and macrophages reached 30–40% confluence in T75 tissue culture flasks, the growth medium (GM) was replaced with 30% TCM in GM (TCM: GM = 3:7) to allow the TCM to induce the fibroblast cells and macrophage cells. For the education, the cells were allowed to grow in the media for 3–4 days then the media was replaced with 3 ml SFM containing with 2% FBS. After 48 h, the supernatant was centrifuged and filtered. The resulting tumor-induced fibroblast and macrophage (MDA-MB-231- fibroblast or macrophage) conditioned media was stored in aliquots at −80°C to avoid multiple freeze-thaws.

0.135 μmol of DMPC, 0.015 μmol of porphyrin-lipid, and in the case of pyE-LN-CO, 0.03 μmol of CO were combined as chloroform solutions in a glass vial. The chloroform was dried under nitrogen and subsequently under vacuum to form lipid films that were then hydrated with 1 mL of PBS. These hydrated films were bath sonicated for 90 min (48 °C for one hour, followed by Bioruptor sonication (low power, 30 second on/off cycles) for 30 min at 40 °C) to generate lipid suspensions. These suspensions were allowed to cool passively to room temperature prior to the drop-wise addition of 0.4 mg mL^–1 apoE3 in a 75 : 1 total lipid : apoE3 molar ratio. The resulting particle suspensions were rotated overnight at 4 °C to allow for gentle mixing, after which free lipid and CO were precipitated via centrifugation at 4 °C, 17 000g for 5 min. The resulting supernatants were filtered through Corning® 0.2 μm syringe filters. PyE-LN solutions used for in vitro and in vivo studies were concentrated by 3 and 5–6-fold respectively using 10 000 NMWL regenerated cellulose Amicon® Ultra-15 centrifuge filter units prior to sterile filtration through Corning® 0.2 μm syringe filters. Particles were shielded from light and stored at 4 °C prior to use.

Ovary pairs from prepubertal three-way cross pigs (mixed Yorkshire, Landrace, and Duroc breeds) obtained from a local slaughterhouse, were collected for follicular fluid analysis. Aspirated porcine follicles were divided into three groups: small (1–2 mm), medium (3–7 mm), and large (≥8 mm) (29 (link), 30 (link)). To eliminate debris and blood, the pFF was centrifuged for 20 min at 94 g and 4°C. The supernatant was extracted using a 10 cc disposable syringe (Sofjec; Hwajin Medical, Chungnam, Korea) with a 0.2 μm syringe filter (Corning, NY, USA) and frozen at −80°C. A porcine CCL2/MCP-1 ELISA kit (ES2RB; Invitrogen, Carlsbad, CA, USA) was used to measure the CCL2 concentration in the pFF samples, according to the manufacturer's instructions. The CCL2 concentration was determined using the mean value of four ELISA experiments, performed in duplicates.

Nonspecific control siRNA (siNS, D-001810-10) and human ELK3 siRNA (siELK3, L-010320-00-0005) were obtained from Dharmacon, Inc. (Chicago, IL, USA). siNS or siELK3 was transfected into LECs using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions. To prepare LEC-conditioned medium (LCM), the complete culture medium from LECs was replaced with serum-free medium (SFM, EBM-2) 24 h after siRNA transfection. The culture medium was collected 24 h after medium replacement and filtered through a 0.2 μm syringe filter (Corning, Tewksbury, MA, USA). LCM was stored in aliquots at −80 °C.