Fibronectin coated 96 well plate

AIC- and VIC-CD151^high/low CMs were sorted and then seeded at 1 × 10⁵ cells/well on a fibronectin-coated 96-well plate (Corning). The cells were fixed after 5–7 d of culture for 20 min with 4% paraformaldehyde, permeabilized for 15 min with 0.1% TritonX100-PBS, and then blocked for 1 h at room temperature with 2% goat serum/0.1% TritonX100-PBS. The fixed cells were stained using anti-MLC-2A (Synaptic systems, 1:100) and anti-MLC-2V (Proteintech, 1:200) overnight at 4 °C. The following day, the cells were washed twice with PBS and stained using Alexa Fluor 647 goat anti-mouse IgG (Thermo Fisher Scientific, 1:500), Alexa Fluor 647 goat anti-rabbit IgG (Thermo Fisher Scientific, 1:500), or Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher Scientific, 1:500) as the secondary antibody for 1 h at 4 °C under dark conditions. The cells were washed twice with PBS and stained with Hoechst (DOJINDO) for 5 min at room temperature. Images were captured using a BZ-X710 (KEYENCE) with a 20× objective.