Cell Culture Techniques 程洪强，方瑜，孙明姣 2013 年 12 月

Cell Thawing and Transfection

Cell Thawing 1.Transfer cryovials containing frozen cells from -80 ℃ to a 37 ℃ water bath. 2.Hold the cryovial on the surface of the water bath with an occasional gentle “flick” during thawing. 3.Dry off the outside of the cryovials and wipe with a 70% ethanol solution before opening the vial to prevent contamination. 4.Transfer the contents of one vial of cells to a 100 mm plate containing 10 ml of medium. 5.Incubate the cells at 37 ℃ /5% CO2 overnight

Cell Transfection 1.Plate cells so they will be 70–90% confluent at the time of transfection. Change the medium with DMEM. 2.Prepare plasmid DNA-lipid complexes. A, 2 ug DNA in 150 ul DMEM B, 10 ul Lipo2000 in 150 ul DMEM (5 min) C, Mix A and B (another 20 min) 3.Add DNA-lipid complexes to cells. 4.After 4-6 hr, remove media and replace with fresh media. 5.Observation GFP signal 24 hr later.