Reducing sds page gels

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.

Flow cytometry was performed on a FACS Canto II (BD Biosciences) and analyzed using FlowJo Software (Treestar). Cells were stained with antibodies to CD3, CD4 (OKT4), CXCR4 (12G5), VLA-4 (9F10) and CD15s (CSLEX1) from BD biosciences, and HECA452, CD45 (2D1), CD44 (BJ14), CD162 (KPL-1), CD43 (CD43-10G7), and FoxP3 (206D) from Biolegend. Staining with mouse E-selectin Ig chimera (RnD Systems) was detected with either anti-His FITC (Bethyl Laboratories) for flow cytometry or secondary rat anti-mouse CD62E (BBIG-E4, RnD Systems) followed by goat anti-rat IgG HRP (Southern Biotech) for western analyses.
Lysates were made from cells by sonication/vortexing in buffer containing 50 mM Tris, 150 mM NaCl, 20 ug/ml phenylmethanesulfonyl fluoride (PMSF), 0.2% NaN₃, protease inhibitor cocktail (Roche), 2% NP40, and 0.2% SDS. Precleared lysates were incubated antibodies to CD43 (1G10, L60, BD Biosciences; 20819, Santa Cruz Biotechnology), PSGL-1 (KPL-1, BD Biosciences; 20929, Santa Cruz Biotechnology), or CD44 (515, BD Biosciences; 2C5, RnD systems). Protein was immunoprecipitated with Protein-G agarose (Life Technologies). Western Blots were run with Reducing SDS-PAGE gels (Bio-Rad).