To begin, place freshly prepared prewarm Sf1Ep cell medium and thawed Sf1Ep cells in a cryo vial on a working platform. Add one milliliter of Sf1Ep cell medium to the cryo vial, and mix gently. Transfer the mixture to a sterile 15-milliliter conical centrifuge tube containing five milliliters of Sf1Ep cell medium, and gently mix.
Centrifuge the cell suspension at 100 G for seven minutes and discard the supernatant before resuspending the pellet in 15 milliliters of fresh Sf1Ep cell medium. Transfer the cell suspension to a T75 tissue culture flask and place it in a standard humidified tissue culture incubator for one week. After incubation, aspirate the medium from the flask and rinse the cell layer with five milliliters of sterile PBS.
After aspirating the PBS, add 2.5 milliliters of trypsin-EDTA to the flask and seal the cap. Incubate the flask at 37 degrees Celsius for five minutes. Tap the flask gently to dislodge the cells and observe under an inverted microscope to confirm the dispersal of the cells.
Add five milliliters of Sf1Ep growth medium and rock the flask to mix with the trypsin-EDTA. Transfer the cell suspension to a conical tube and quantitate the cells using a hemocytometer or automated cell counter. To freeze Sf1Ep cells, spin the cells at 100 G for seven minutes and resuspend the pellet in Sf1Ep medium supplemented with 10%DMSO.
Distribute one milliliter of cell suspension to each cryo vial and freeze overnight at minus 70 to minus 80 degrees Celsius.
