ECM Suspension Preparation: A Technique to Obtain Homogenous Suspension from Tissue-derived Extracellular Matrix
Transcrição
Extracellular matrix or ECM, a complex support network in tissues, consists of proteoglycans and proteins.
To obtain homogenized ECM, begin by taking a measured amount of decellularized ECM powder in a tube. Now, add an acidified buffer to the tube to form an ECM suspension.
Simultaneously, in another tube, dissolve alpha-amylase – a glycolytic enzyme – in the same acidified buffer. The acidic pH of this buffer facilitates the optimal activity of the alpha-amylase enzyme.
Next, supplement the tube containing ECM suspension with alpha-amylase enzyme solution. Incubate the tube with constant agitation. Alpha-amylase hydrolyses the glycans – the carbohydrate moieties – present in the proteoglycans.
Centrifuge to pellet the protein-rich ECM fraction, leaving the digested glycans and spent enzymes in the supernatant. Discard the supernatant.
Resuspend the pellet in sodium chloride solution. The chloride ions extract the proteins from the ECM pellet into the suspension. Centrifuge and discard the sodium chloride-containing supernatant.
Next, add acetic acid to the protein-containing ECM pellet. Acetic acid further solubilizes ECM protein aggregates and brings them in suspension.
Use a homogenizer to obtain a uniform ECM suspension. Store the homogeneous ECM suspension at 4 °C for further downstream analysis.
