CellCultureandCellIsolation.pptVIP

* * Cell Culture and Cell Isolation Basic Techniques for Mammalian Cell Tissue Culture Cell Isolation Ruijun Zhang Institute of Immunology in USTC Basic Techniques for Mammalian Cell Tissue Culture Cell subculturing Cell freezing Cell recovering Cell counting Cell subculturing Trypsinizing and Subculturing Cells From a Monolayer Passaging Cells in Suspension Cultures PROCESS Remove all medium Washing cells with Ca2+ Mg2+-free PBS Trypsin/EDTA solution to dislodge cells Wash cells and count the cells number Complete medium to inhibit the Trypsin activity Add fresh medium to culture cells Note Ca2+ and Mg2+ in the salt solution can cause cells to stick together and inhibit the trypsin activity Inhibit the further trypsin activity to protect the cells PROCESS Swirl flask to suspend the cells Remove cell suspension Add fresh medium to culture cells Split the cells Washing cells Cell freezing The process The note PROCESS Harvest the cells with trypsin Washing cells Resuspend the cells with freezing medium Transfer the cells into liquid nitrogen storage freezer Adjust the cells concentration of 106~107/ml Gradual temperature drop Harvest the cells Cells from suspension Cells from monolayer Note Freezing medium: complete medium supplemented with 30% -50%(v/v) FBS and 10% (v/v) DMSO Gradual temperature drop Keep out to be contaminated Be sure to suspend the cell pellet carefully Cell recovering The process The note PROCESS Remove vial from liquid nitrogen freezer Immediately place it into a 37°C water bath Transfer cells into a sterile centrifuge tube Check cultures after 24h Washing cells Add fresh medium to cell pellet and culture Cells from suspension Cells from monolayer Note Care should be taken not to spill liquid nitrogen on the skin. The medium should be thawed as quickly as possible. Alternative temperature 42?C. Cell counting The process Cell activi