A1r mp confocal microscopy

Mdx mice were purchased from the Jackson Laboratory (stock #001801). Mice were housed in the animal facility with free access to water and standard rodent chow. All procedures involving the use of animals were performed in accordance with the guidelines presented by Purdue University's Animal Care and Use Committee. Without special mentioning, we used 2-month-old male mice for experiments. Cardiotoxin (CTX) injection (10 μM) was administered in the tibialis anterior (TA) muscle of mdx mice 24 h prior scaffold implantation. 2 × 10⁵ primary myoblasts were seeded on Matrix 3 fibers and cultured overnight in growth media. Scaffolds (2 mm in diameter) were implanted at the site of CTX administration. Myoblasts cells (2 × 10⁵ cells in 500 μL primary myoblast growth medium) were injected at the site of CTX administration and were used as controls. The TA muscle, for both control and scaffold group, was harvested by cryo-fixing the samples in OCT 3 weeks after implantation. Cryosections of the TA muscles were stained for dystrophin (ab15277, Abcam; 1:1000 dilution) for further analysis. The stained cross-sections (230 × 230 μm) were imaged in Nikon A1R MP confocal microscopy and quantified to evaluate dystrophin positive myofibers per cross section image obtained (n = 3 per group).

To visualize uptake of nanoparticles by NK cells, NK‐92 cells were seeded in the wells of a 24‐well plate at the density of 5.0 × 10⁴ per well and 500 µL of nanoparticle suspension (in ddH₂O, ≈5 mg mL⁻¹) was added into the wells. Free FITC (in PBS) solution was prepared as a control. After incubation at 37 °C for 3 h, the cells were collected by centrifugation at 125 × g for 10 min, washed with 1× PBS three times and stained with PI (5 µg mL⁻¹) for 5 min at room temperature. After washing excess stain off, the cells were transferred into a 35 mm glass‐bottom dish for imaging by Nikon A1RMP confocal microscopy.