Altman:Protocols/TC Protocols/Freezing SF9

Department of Physics, Willamette University

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Freezing down SF9 cells for long-term storage

1) Grow the cells to a density that is greater than or equal to 1*10^7 viable cells/mL

2) Pellet the cells by centrifugation: place the 35 mL of cells in a 50-mL conical, and spin at 1000 rpm for 10 minutes

3) Remove the media; this media is "conditioned media"

4) Combine the conditioned media 1:1 with fresh media

5) Add DMSO to the media to a final concentration of 7.5% (e.g. 2.6 mL DMSO + 35 mL media)

6) Resuspend the cells in the media+DMSO to return the cells to a final density of greater than or equal to 1*10^7 viable cells/mL

7) Aliquot 1.5 mL of cells into 2.0 mL cryovials

8) Place tubes between two pieces of styrofoam and place in a -20 freezer overnight

9) Place in liquid nitrogen for long term storage