Quantifying Extracellular Matrix Proteins in ASCs

The retention of fibronectin and laminin in non-/decellularization ASCs was measured by Western blot analysis as described previously [14 (link)]. Briefly, the sample lysates were prepared in RIPA buffer containing protease and phosphatase inhibitor cocktail (all from Thermo Fisher Scientific) as well as 1 mM phenylmethylsulfonyl fluoride (Calbiochem). Protein concentrations were measured using a BCA kit (Thermo Fisher Scientific), and equal amounts of protein samples were loaded onto reducing SDS-PAGE gels (Bio-Rad), separated by electrophoresis, and then transferred to PVDF membranes (Bio-Rad). After blocking with 5% skim milk, membranes were incubated overnight (4°C) with the following primary antibodies: anti-fibronectin (1 : 1000, Abcam), anti-laminin (1 : 1000; Abcam), and anti-GAPDH (1 : 2000 Abcam). Then the membranes were washed 3 times with PBST before following incubation with species-specific HRP-conjugated secondary antibodies. After another 3 washes with PBST, chemiluminescence detection was performed using an ECL kit (Thermo Fisher Scientific, USA). GAPDH served as internal controls to confirm that decellularized matrices did not contain cell-associated residuals.