TissueCulture:Thawing cells: Difference between revisions
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*10-50 ml centrifuge tube | *10-50 ml centrifuge tube | ||
*Cell culture medium (depends on cell line/cells) | *Cell culture medium (depends on cell line/cells) | ||
*Trypsin-EDTA (e.g. from [http://www.paa.com PAA], L11-004) - for adeherent cells | *Trypsin-EDTA (e.g. from [http://www.paa.com PAA], L11-004) - for adeherent cells | ||
*Basic TC centriguge (0 - 2500 rpm) | *Basic TC centriguge (0 - 2500 rpm) | ||
==Thawing== | ==Thawing== | ||
#Keep frozen cells on dry ice until ready for thawing. | # Setup two vials of cold (4 C) media (10 ml) in a 15 ml tube | ||
#Swirl | # Warm remaining media (~5-8 ml) in a 15 ml tube to 37C | ||
# | # When ready to thaw, remove vial of cells from liquid nitrogen | ||
# | #:Keep frozen cells on dry ice until ready for thawing. | ||
# | # Swirl the vial in the water bath (37C) until a small ice chip is left in the tube (~1 min) | ||
# | # Place the vial in the hood and clean it with 70% ethanol | ||
# | # Immediately remove the contents of the vial and place into the cold media | ||
# | # Rinse the tube down with some of the cold media from the second vial | ||
# Spin down cells at 2000 rpm (80-100 x g) for 5 min | |||
# Aspirate off the cold media and resuspend the cells in the warm media | |||
# Transfer the cells and the media into a petri dish | |||
# Count cells | |||
# Place in incubator | |||
# Change the media once the cells have attached to the plate | |||
#: Removes remaining DMSO | |||
# Allow cells to grow to 80-90% confluency | |||
# Split cells (1:3) into petri dishes | |||
# Continue to split cells and freeze down as needed | |||
==Notes== | ==Notes== | ||
Line 50: | Line 35: | ||
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites. | #You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites. | ||
#Anecdotal observations that might be of use to others can also be posted here. | #Anecdotal observations that might be of use to others can also be posted here. | ||
'''Alternative Thawing''' | |||
* From step 5 above, using a 1 ml pipet add 1 ml of the cold media from the 15 ml tube drop by drop to the vial (about 1 drop every 10 seconds). | |||
* Plate immediately with the cold media and change the media once cells have attached (12 - 24 hours later) to remove DMSO that was in the freezing media. Usually a 1:3 ratio is good for MC3T3s and a 1:4 ratio for MLOY4s. | |||
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | ||
== | |||
See also | |||
:[[Jacobs:Protocol_Freezing_and_Thawing_Cells]] | |||
:[[WangLab:Thawing_Cells]] | |||
:[[Marek:_Freeze-down/Thaw]] | |||
:[[Wittrup:_Thawing_Protocol]] | |||
==Contact== | ==Contact== |
Latest revision as of 23:14, 24 May 2012
Overview
This short protocol describes how to freeze down and thaw (or bring up) mammalian cells (e.g. HeLa, KEK293, CHO, Jurkat T cells).
Materials
List reagents, supplies and equipment necessary to perform the protocol here.
- 10-50 ml centrifuge tube
- Cell culture medium (depends on cell line/cells)
- Trypsin-EDTA (e.g. from PAA, L11-004) - for adeherent cells
- Basic TC centriguge (0 - 2500 rpm)
Thawing
- Setup two vials of cold (4 C) media (10 ml) in a 15 ml tube
- Warm remaining media (~5-8 ml) in a 15 ml tube to 37C
- When ready to thaw, remove vial of cells from liquid nitrogen
- Keep frozen cells on dry ice until ready for thawing.
- Swirl the vial in the water bath (37C) until a small ice chip is left in the tube (~1 min)
- Place the vial in the hood and clean it with 70% ethanol
- Immediately remove the contents of the vial and place into the cold media
- Rinse the tube down with some of the cold media from the second vial
- Spin down cells at 2000 rpm (80-100 x g) for 5 min
- Aspirate off the cold media and resuspend the cells in the warm media
- Transfer the cells and the media into a petri dish
- Count cells
- Place in incubator
- Change the media once the cells have attached to the plate
- Removes remaining DMSO
- Allow cells to grow to 80-90% confluency
- Split cells (1:3) into petri dishes
- Continue to split cells and freeze down as needed
Notes
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Alternative Thawing
- From step 5 above, using a 1 ml pipet add 1 ml of the cold media from the 15 ml tube drop by drop to the vial (about 1 drop every 10 seconds).
- Plate immediately with the cold media and change the media once cells have attached (12 - 24 hours later) to remove DMSO that was in the freezing media. Usually a 1:3 ratio is good for MC3T3s and a 1:4 ratio for MLOY4s.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
== See also
- Jacobs:Protocol_Freezing_and_Thawing_Cells
- WangLab:Thawing_Cells
- Marek:_Freeze-down/Thaw
- Wittrup:_Thawing_Protocol
Contact
- Who has experience with this protocol?
or instead, discuss this protocol.