Bacmam gfp transduction control

BacMam GFP Transduction Control (Invitrogen) was added to ADSC culture medium (150 μL/12 mL culture medium, i.e., approximately 1.5 × 10⁶ cells). Incubated for 10 min at room temperature and then left overnight in ADSC culture. After this time the BacMam GFP medium was removed and the culture was supplemented with a fresh medium. The procedure was carried out as recommended, based on the manufacturer’s protocol.

We investigated the surface binding and internalization of our anti-CD276 mAb using live-cell confocal imaging following established protocols in our previous publications [29 (link),30 (link),33 (link)]. H460-Fluc and human NSCLC were cultured in 35 mm glass bottom dishes (Cellvis, Mountain View, CA, USA) at a density of 1 × 10⁴ cells per dish in 1.5 mL of medium. To visualize the cytoplasm and nucleus, BacMam GFP Transduction Control (Invitrogen) and NucBlue™ Live ReadyProbes™ Reagent (Invitrogen), respectively, were used for staining following manufacturing protocols. Next, the AF647-labeled CD276 mAb was added to the cells at a final concentration of 1 or 5 µg per mL. Live-cell images were captured at 0–24 h after the addition of CD276 mAb-AF647 using a Nikon A1R-HD25 confocal microscope (Nikon USA, Melville, NY, USA). This experimental approach allowed us to dynamically monitor the binding and internalization of CD276 mAb into NSCLC over different time points.

For co-culture experiments, 25 × 10³ mouse C2C12 myoblasts (Sigma–Aldrich, St. Louis, MO, USA) and 25 × 10³ of rADSCs were seeded on sterile coverslips and cultured in high-glucose DMEM supplemented with 10% of FBS and 10 μg/mL gentamycin, in the presence or absence of IL-4 or SDF-1 for 7 days. Rat ADSCs used for the co-culture were first labelled with BacMam GFP Transduction Control (Invitrogen), according to the manufacturer’s instructions. Culture medium was replaced every day to keep required levels of cytokines. Cells were fixed with 3% paraformaldehyde (PFA) and subjected to immunolocalizatio analysis. BacMam GFP allowed for the identification of hybrid myotubes, i.e., ones generated by rADSCs fusing with C2C12 myoblasts. Nuclei were stained with Hoechst 33,342 (Sigma Aldrich) or DRAQ5 (Biostatus Limited), diluted 1:1000 in PBS. In selected experiments, cells were stained with either anti-myosin (skeletal) antibody (M7523, Sigma Aldrich) or anti-skeletal muscle marker (12/101-c, DSHB) to visualize the localization of myotubes in the culture. For each experimental group, the number of hybrid myotubes was counted from at least five fields of view of three independent experiments.