cell anking related protocol summary.docVIP

A. Cell culture 1. Cell thawing Remove tubes from liquid nitrogen and thaw the vials by gentle agitation in a 37oC water bath (Thawing should be rapid in approximately 2 minutes). To reduce the possibility of contamination, keep the O-ring and cap out of the water. Remove the vials from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spaying with 70% alcohol (All of the operations from this point on should be carried out under strict aseptic conditions). Transfer the vial contents to a centrifuge tube containing 9.0ml complete culture medium and spin at at 1000rpm for 10min. Remove supernatant, then resuspend the cell pellet with 4ml complete culture medium and dispense into a 6cm dish. Incubate the culture at 37oC in 5% CO2 incubator (No CO2 is recommended only if the medium is L-15). 2. Cell sub-culturing Cells are checked microscopically to ensure they are healthy and growing as expected. For attached cell lines, refer to 4 to 7. For suspension cell lines, refer to 8 to 10. Remove and discard culture medium using a vacuum pump. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.038% (w/v) EDTA solution to remove all traces of serum that contains trypsin inhibitor. Add appropriate volume of trypsin-EDTA solution and observe cells under an inverted microscope until cell layer is dispersed. Add some complete growth medium and aspirate cells by gently pipetting. Cultures can be maintained by addition of fresh medium. Alternatively, transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 10 minutes. Discard the supernatant using a vacuum pump. Add appropriate volume of complete medium. Suspend the cell pellet by gently pipetting. Adjust the density of the culture and maintain it as recommended. 3. Cell freezing Before cryopreservation, cells should be characterized and checked for contamination. Cells are checked microscopically to ensure they are healthy and make sure cells are in log phase