To Image The Cytoskeleton of Cells Proliferation on Biomaterial Surface.
Our research group focuses on the fabrication and designing of biomaterials for hard tissue (bone) replacement. The biocompatibility of these biomaterials is tested by performing cell culture studies (adhesion, growth and proliferation) on biomaterial. Examination of the cell morphology is conducted by using fluorescence microscopy technique. Here, in this experiment, imaging of the cytoskeleton of the cells is performed by using a specific dye or a fluorophore which binds to the actin subunits of the cytoskeleton and then fluoresces green under fluorescence microscope.
(1) Clean and sterilize the specimen. (2) Perform the cell culture or other experiment on the specimen for the desired period. (3) Dehydrate the specimen to stop the motion of the cells in case of cell culture. (4) Fix the specimen using glutaraldehyde. (5) Wash in PBS. (6) Treat it with Triton and then again wash in PBS. (7) Treat the specimen with the phalloidin dye. (8) Place the suitable filter in the optical axis and turn on the mercury-vapor lamp. (9) Adjust the intensity of the excitation light. (10) Keep the specimen under the fluorescence microscope. (11) Focus the desired region using eyepiece to view the cytoskeleton of the specimen. (12) Capture the digital images using digital camera for phalloidin dye.