Beadbug 3 place microtube homogenizer

Tumors dissected from NOD-SCID mice and PyMT-MMTV mice and mammary fat pads from C57BL/6 mice were submerged in 0.1% SDS solution and stirred for several days, replacing the solution twice daily. Once the tissue turned completely white (visual sign of successful decellularization), the solution was replaced with a 0.05% Triton X-100 solution for 3 hours. The tissues were then washed for 4 days by stirring in dH₂O to remove any residual detergent. Decellularized tissue was sectioned and H&E-stained, along with Western blot, to assess decellularization. The dECM or intact tissues were lysed in 25 mM tris, 150 mM NaCl, 10% glycerol, 1% NP-40, and 0.5 M EDTA with 1× protease Mini-complete protease inhibitor (04693124001; Roche, Indianapolis, IN) and 1× phosphatase inhibitor cocktail (4906845001; Roche, Indianapolis, IN) at 4°C. In addition, for dECM, tissue homogenization was performed using the BeadBug 3 Place Microtube Homogenizer (D1030; Benchmark Scientific, Sayreville, NJ). The dECM or cell homogenate was then spun down at 21,000g for 10 min at 4°C, and the supernatant was stored at −20°C until used for Western blot or proteomics analysis.

dECM, intact tumors, or cell pellets were lysed in 25 mmol/L tris, 150 mmol/L NaCl, 10% glycerol, 1% NP-40, and 0.5 mol/L EDTA with 1× protease Mini-complete protease inhibitor (04693124001; Roche) and 1× phosphatase inhibitor cocktail (4906845001; Roche) at 4°C. In the case of dECM and intact tumors, mechanical homogenization was achieved using the Bead-Bug 3 Place Microtube Homogenizer (D1030; Benchmark Scientific). The resulting homogenate was then centrifuged at 21,000 g for 10 minutes at 4°C and the supernatant was stored at −20°C for subsequent analysis.