# Altman:Protocols/TC Protocols/Freezing SF9

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 Revision as of 15:42, 24 July 2013 (view source) (New page: {{Template:Altman}}
==Freezing down SF9 cells for long-term storage== : 1) Grow the cells to a density that is grea...)← Previous diff Revision as of 15:44, 24 July 2013 (view source) (→Freezing down SF9 cells for long-term storage)Next diff → Line 5: Line 5: ==Freezing down SF9 cells for long-term storage== ==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 + 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" + 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 - : 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) + 3. Remove the media; this media is "conditioned 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 + 4. Combine the conditioned media 1:1 with fresh media - : 8) Place tubes between two pieces of styrofoam and place in a -20 freezer overnight + - : 9) Place in liquid nitrogen for long term storage + 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

## Revision as of 15:44, 24 July 2013

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