Jacobs:Protocol Freezing and Thawing Cells: Difference between revisions
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==Procedure== | ==Procedure== | ||
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* Trypsin | * Trypsin | ||
* Trypan Blue | * Trypan Blue Solution (0.4%) | ||
* Hemocytometer | * Hemocytometer | ||
* Media for frozen cells | * Media for frozen cells | ||
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# Check cell culture log and be sure there is room for frozen vials | # Check cell culture log and be sure there is room for frozen vials | ||
# Label vials with cell type, date, passage number, etc. | # Label vials with cell type, date, passage number, etc. | ||
# Collect cell suspension by trypsinizing | # Collect cell suspension by trypsinizing (Take out media and put in 3 mL of trypsin) | ||
# Combine all flasks into 50 ml tube | # Combine all flasks into 50 ml tube | ||
# Count cells | # Count cells | ||
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## 1-2 million cells per vial for MLO-A5 | ## 1-2 million cells per vial for MLO-A5 | ||
# Make fresh freezing down media | # Make fresh freezing down media | ||
## General: 95% FBS + 5% DMSO | ## General (including FAK cells): 95% FBS + 5% DMSO | ||
## MLOs: 50% α-MEM, 40% FBS, 5%DMSO | ## MLOs: 50% α-MEM, 40% FBS, 5%DMSO | ||
# Spin down the cell suspension and pellet the cells (1500rpm, 7-10 min) | # Spin down the cell suspension and pellet the cells (1500rpm, 7-10 min) | ||
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* Media for cells | * Media for cells | ||
* Petri dishes for cells | * Petri dishes for cells | ||
* Trypan Blue | * Trypan Blue Solution (0.4%) | ||
* Hemocytometer | * Hemocytometer | ||
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# Split cells (1:3) into petri dishes | # Split cells (1:3) into petri dishes | ||
# Continue to split cells and freeze down as needed | # Continue to split cells and freeze down as needed | ||
'''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. | |||
==References== | ==References== | ||
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==Contact== | ==Contact== | ||
*History: CMBL – CRJ/JJR, last updated | *History: CMBL – CRJ/JJR, last updated 2/17/11 by AMN | ||
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | ||
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[[Category:In vitro]] | [[Category:In vitro]] | ||
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Latest revision as of 13:44, 10 August 2011
Procedure
Materials for Freezing
- Trypsin
- Trypan Blue Solution (0.4%)
- Hemocytometer
- Media for frozen cells
- DMSO
- Gloves, 70% ethanol, etc.
Freezing
- Check cell culture log and be sure there is room for frozen vials
- Label vials with cell type, date, passage number, etc.
- Collect cell suspension by trypsinizing (Take out media and put in 3 mL of trypsin)
- Combine all flasks into 50 ml tube
- Count cells
- 1 million cells per vial (for MC=4 vials/flasks, for UMR=6 vials per flask.)
- 1-2 million cells per vial for MLO-A5
- Make fresh freezing down media
- General (including FAK cells): 95% FBS + 5% DMSO
- MLOs: 50% α-MEM, 40% FBS, 5%DMSO
- Spin down the cell suspension and pellet the cells (1500rpm, 7-10 min)
- Aspirate media
- Resuspend cell pellet in freezing media
- Aliquot 1ml freezing media per 2ml tube
- Place tubes in round isopropanol box in -80C freezer OR
- Transfer to the -20C for 2-3 hours
- Transfer to the -80C for at least overnight
- Store permanently in liquid nitrogen within 1-2 days
- Be sure to record in the cell culture log where the cells are permanently stored
Materials for Thawing
- Media for cells
- Petri dishes for cells
- Trypan Blue Solution (0.4%)
- Hemocytometer
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
- 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 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
- Allow cells to grow to 80-90% confluency
- Split cells (1:3) into petri dishes
- Continue to split cells and freeze down as needed
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.
References
Contact
- History: CMBL – CRJ/JJR, last updated 2/17/11 by AMN
or instead, discuss this protocol.
