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Cell Culture Techniques

Cell Culture Techniques. 程洪强，方瑜，孙明姣 2013 年 12 月. Cell Thawing and Transfection. Cell Thawing. Transfer cryovials containing frozen cells from -80℃ to a 37 ℃ water bath. Hold the cryovial on the surface of the water bath with an occasional gentle “flick” during thawing.

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Cell Culture Techniques

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

  2. Cell Thawing and Transfection

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

  4. Cell Transfection • Plate cells so they will be 70–90% confluent at the time of transfection. Change the medium with DMEM. • 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) • Add DNA-lipid complexes to cells. • After 4-6 hr, remove media and replace with fresh media. • Observation GFP signal 24 hr later.

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