Altman:Protocols/TC Protocols/Freezing SF9: Difference between revisions

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(New page: {{Template:Altman}} <div style="padding: 10px; width: 700px; border: 5px solid #B22222;"> ==Freezing down SF9 cells for long-term storage== : 1) Grow the cells to a density that is grea...)
 
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==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 12:44, 24 July 2013


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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

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