ECM Suspension Preparation: A Technique to Obtain Homogenous Suspension from Tissue-derived Extracellular Matrix
Trascrizione
Weigh out 250 milligrams of either minced or cryomilled ECM and transfer it to a 15-milliliter centrifuge tube. Add 5 milliliters of 0.22 molar NaH2PO4 buffer to the centrifuge tube and mark the liquid level on the tube. Then, prepare an alpha-amylase stock solution by adding 7.5 milligrams of alpha-amylase to 1 milliliter of 0.22 molar NaH2PO4 buffer.
Next, add 100 microliters of the alpha-amylase stock solution to the sample. Then, add 0.22 molar NaH2PO4 to obtain a final volume of 10 milliliters. Agitate the suspension continuously at 300 rpm and room temperature for 72 hours.
Centrifuge the suspension at 1,500 x g for 10 minutes. Carefully collect and discard the supernatant without disturbing the digested ECM pellet. Then, use 10 milliliters of 5% NaCl diluted in double distilled water to resuspend the pelleted material and agitate continuously for 10 minutes at 300 rpm.
Repeat the wash two more times before using 10 milliliters of double distilled water to resuspend the pellet. Then, agitate the suspension continuously at 300 rpm at room temperature for 10 minutes. After centrifuging the sample again, carefully collect and discard the supernatant without disturbing the digested ECM pellet.
Add 0.2 molar acetic acid to the 5-milliliter mark and vortex. Then, continuously agitate the suspension at 120 rpm and 37 degrees Celsius overnight. The following day, using a hand-held homogenizer equipped with a 10-millimeter wide saw tooth probe, homogenize the ECM suspension at room temperature in 10-second intervals until no visible fragments remain.
