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The project goal is to dissect the mechanical feedback of cells onto the extracellular matrix (ECM) using De Novo ECM synthesis and traction force microscopy (TFM). In this thesis, we devised a method to apply traction force microscopy to De Novo ECM. We first stimulated 3T3s to synthesize ECM (>=20μm in 10days) using ascorbic acid with experimental conditions of hyperglycemia. After cell removal by lysis, the ECM was measured with AFM and fresh cells were replated for TFM. To achieve TFM on de novo ECM, we developed a serial labeling approach to deposit carboxylated fluorescent beads during multi-day ECM synthesis. We observe that ECM stiffness is increased in high vs low glucose. We also observe a unimodal response of cell traction to increasing stiffness in polyacrylamide substitutes. With the generated TFM bead displacement data, we plan to analyze 3t3s replated onto bead-labeled de novo ECM to better understand the mechanical response of cells. |
