细胞迁移和浸润实验操作 protocol.pdfVIP

For life science research only. Not for use in diagnostic procedures. System Technical Note No. 3 / February 2010 General Cell Migration Protocol Using the CIM-Plate 16 Cell Migration Assay Using the CIM-Plate 16 with the RTCA DP Instrument The protocol described below is a basic migration assay protocol for the RTCA DP Instrument using fetal bovine serum (FBS) as a chemoattractant. The protocol is optimized for the HT-1080 human fi brosarcoma cell line and the HeLa human cervical cancer cell line. The assay conditions are subject to further optimization if different types of cell lines or chemoattractants are used. 1. Protocol 1.1 Reagents HeLa and HT-1080 cells, purchased from ATCC and at 60% – 80% confl uence at the time of detachment The ultimate success of the migration experiments is dependent upon cell culture conditions prior to the assay and conditions used for detaching the cells from the fl ask. The number of cells used in a migration experiment will ultimately depend on the cell type being used. It is imperative to conduct preliminary experiments and determine the optimal cell number for each cell line. We recommend initially seeding cells in the range of 10,000 – 80,000 cells in a fi nal volume of 100 µl. Trypsin-EDTA solution for cell detachment, or Non-enzymatic Cell Dissociation Solution (Sigma Cat. No. C5789) for cells that may be especially sensitive to enzymatic methods of cell detachment If using the protease method for cell detachment, it is important to minimize the time of incubation with the detachment solution. Cell surface receptors (such as integrins) play an important role in cell migration and it is important to conserve the number and integrity of these receptors on the cell s