Ultracel 10 membrane

Samples were centrifuged at 16,000× g for 60 min at 4 °C using the centrifugal filter (Merck Millipore, Darmstadt, Germany, Microcon, −10 kDa with Ultracel-10 membrane). After centrifugation, the filtered solution was analyzed to determine drug concentration using the UV–VIS spectrophotometry (Thermo Fisher Scientific, 60S Evolution, Madison, WI, USA) method at 239 nm, utilizing the equation from the fitted standard curve plot constructed previously [24 (link)]. The encapsulation efficiency (EE) was calculated by using Equation (1), where the total drug amount added (Drug_total) and the non-entrapped drug (Drug_free) in the filtered solution were considered [36 (link)].
$\mathrm{EE} (\%)=\frac{\left[\mathrm{Drug}\right]\mathrm{total}-\left[\mathrm{Drug}\right]\mathrm{free}}{\left[\mathrm{Drug}\right]\mathrm{total}}\times 100$

2×10⁵ LAM patient-derived cells or TSC2-reexpressing cells were seeded in 6-well plate with 2 mL IIA complete medium supplemented with 10% FBS. When cells became 70% confluent, medium was replaced with IIA serum-free (0% FBS). 24 hr later, medium was collected and centrifuged at 2,000 rpm at 4°C for 10 min. Supernatant was transferred to Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-10 membrane (10 kDa cutoff) (EMD Millipore) and concentrated.

After overnight treatment with reagents in phenol red-free DMEM (Fujifilm Wako Pure Chemical Corporation), the cell culture medium was collected and centrifuged at 1000 × g for 15 min, and the supernatant was used as a sample solution. At 16 h after the intraperitoneal injection of reagents, blood was taken from the mouse hearts. Then, the blood was centrifuged at 10,000 × g for 2 min to separate the serum using a BD Microtainer Tube (Becton, Dickinson and Company, NJ, USA). The serum was centrifuged with an Amicon Ultra4 Centrifugal Filter Unit with an Ultracel-10 membrane (Merck, Darmstadt, Germany) at 14,000 × g for 15 min to remove proteins, and the protein-free serum was used as a sample solution. The combined concentrations of NO₂⁻ and NO₃⁻, which are the degradation products of NO, were measured using a NO₂/NO₃ Assay Kit (Dojindo Laboratories, Kumamoto, Japan) and a plate reader system (FlexStation 3, Molecular Devices, CA, USA). Total NO₂⁻/NO₃⁻ production indicates NO production.

Hemoglobin (10 µmol/L) was oxidized with H₂O₂ (40 µmol/L) at room temperature for 30 min. To assess the effect of haptoglobin on the fragmentation of hemoglobin, hemoglobin was incubated with haptoglobin (Hp 1-1, 330-12, Lee BioSolutions, 10 µmol/L) and then exposed to H₂O₂ (40 µmol/L) at room temperature. The oxidized products were concentrated with an Amicon Ultra-0.5 centrifugal filter unit with an Ultracel-10 membrane (UFC501096, Merck KGaA, Darmstadt, Germany). The concentrated fraction (containing 30 µg of protein) was subjected to SDS-PAGE (NW04120BOX, NuPAGE Bis-Tris Precast Gel, Thermo Scientific).

EVs were isolated using Cushioned-Density Gradient Ultracentrifugation (C-DGUG) [53 (link),54 (link)]. Briefly, ~40 mL of the serum/antibiotic-free media was centrifuged at 3000× g for 15 min to eliminate cell debris, followed by filtration (0.22 µm). The filtered media were added to a liquid cushion (60% Iodixanol) and ultracentrifuged at 150,000× g for 4 h at 4 °C to concentrate the small vesicles. Subsequently, these vesicles were resolved in a density gradient (40, 20, 10, and 5% iodixanol) and submitted to ultracentrifugation at 100,000× g for 18 h at 4 °C. The EV-rich, protein-low fractions (6 and 7) were then pooled and concentrated to 500 µL on an Amicon Ultra-4 Centrifugal Filter Unit with an Ultracel-10 membrane (MWCO = 10 kDa; Merck Millipore, Darmstadt, Germany, United States Affiliate) by centrifugation at 4000× g for 30 min.