2.15. Fluorescent Micelle Loaded Macrophage-RASMC Co-Culture

Synchronized RASMC (seeded at 5 × 103 cells in 96 well glass bottom imaging plate, 655891, Greiner Bio-One, Monroe, NC, USA) were treated with DiD micelle-loaded and empty RAW 264.7 macrophages at a ratio of 1:10 RASMC to macrophages. Macrophages were pretreated with 5 µM DiD or DiD-ARAPas for 2 h. Control RASMC were treated with 250 nM of DiD, which is equivalent to the amount of DiD loaded in 5 × 10⁴ macrophages. Cells were co-cultured in phenol-free media 1:1 high glucose DMEM:F12 (21041025, Gibco, Gaithersburg, MA, USA). Cells were imaged every hour for 24 h using a cell imaging multi-mode reader (Cytation 5) equipped with a CO₂ chamber. For confocal microscopy, synchronized RASMC (seeded at 2 × 10⁴ cells in 96-well glass bottom imaging plate, 655891, Greiner Bio-One, Monroe, NC, USA) were treated with DiD-labeled micelle-loaded and also empty RAW 264.7 macrophages at a ratio of 1:10 RASMC to macrophages. Macrophages were pretreated with 5 µM DiD-ARAPas for 2 h. RASMC were also treated with 1 µM of DiD, equivalent to the amount of DiD loaded in 2 × 10⁵ macrophages at the same volume. After 24 h, cells were fixed in 2% paraformaldehyde, and permeabilized with 0.1% Triton-X (X100; Sigma-Aldrich). Cells were then probed for alpha smooth muscle actin (1:200, 48938; Cell Signaling Technology, Danvers, MA, USA) followed by AlexaFluor555 goat anti-mouse IgG (2 µg/mL, A21425; Thermo-Fisher Scientific, Waltham, MA, USA). All antibodies were diluted in IHC-Tek diluent (1W-1000; IHC World, Woodstock, MD, USA); nuclei were counterstained with 0.0012 µM DAPI (D3571; Invitrogen, Carlsbad, CA, USA). Stained samples were maintained and imaged in PBS. Image acquisition was done using a LSM 780 laser scanning confocal microscope (Carl Zeiss Microscopy, Jena, Germany). Three-dimensional reconstructions were carried out using Imaris v9.5.0 (Bitplane AG, Zürich, Switzerland).