TissueCulture:Thawing cells - Revision history

Christopher C Vanlang at 06:14, 25 May 2012

2012-05-25T06:14:03Z

←Older revision		Revision as of 06:14, 25 May 2012
Line 7:		Line 7:

	*10-50 ml centrifuge tube		*10-50 ml centrifuge tube
-	*cryo tubes
-	*DMSO, Tissue culture (TC)-tested (e.g. [http://www.sigmaaldrich.com Sigma], D2650)
	*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
-	*'''Freeze-down medium'''
-	**6-10% DMSO
-	**Cell culture medium (I recommend to use at least 20% FCS and you can use 100% fetal calf serum (FCS) instead of cell culture medium)
	*Basic TC centriguge (0 - 2500 rpm)		*Basic TC centriguge (0 - 2500 rpm)
-	*Freeze-down box (this could be simply made from a small styropore box and cotton or from two styropore racks from 15-ml centrifuge tubes )
-
-	~~==Freeze-down (suspension cells)==~~
-	~~#Centrifuge at 80-100 x g for 4-5 min at RT.~~
-	~~#Suck out the medium.~~
-	~~#Resuspend 1-5 x 10*6 cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).~~
-	~~#Transfer cryo-vials with cells into freeze-down box and store at -80 gr.C.~~
-	*Leave over-night at -80 gr.C.
-	*You can store cells at -80 gr.C for up to 6 months.
-	*Transfer cells into liquid nitrogen a day later for longer storage (1-20 years or so).
-
-	~~==Freeze-down (adherent cells)==~~
-	~~#Wash cells with PBS.~~
-	~~#Add thin layer of Trypsin-EDTA on cells (approx. 1ml for 90 mm Petri dish)~~
-	~~#Observe cells under microscope until these are rounded.~~
-	~~#Add serum-containing medium and resuspend cells. Transfer into centrifuge tubes.~~
-	~~#Centrifuge at 1000-1200 rpm for 4-5 min at RT.~~
-	~~#Suck out the medium.~~
-	~~#Resuspend cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).~~
-	~~#Transfer cells into freeze-down box and place at -80 gr.C. (Comments as for suspension cells).~~

	==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 a vial ~~with frozen cells~~ in ~~37gr.C~~ water bath ~~and remove just while~~ a ~~sliver of~~ ice ~~remains.~~	+	# Warm remaining media (~5-8 ml) in a 15 ml tube to 37C
-	#~~Spray tube~~ with 70% ethanol ~~for sterilization.~~	+	# When ready to thaw, remove vial of cells from liquid nitrogen
-	#~~Aspirate cells from cryo-tube~~ into ~~pre-half-filled pipette with warm culture medium.~~	+	#:Keep frozen cells on dry ice until ready for thawing.
-	#~~Fill centrifugation~~ tube with ~~culture medium: at least 10 ml total volume.~~	+	# Swirl the vial in the water bath (37C) until a small ice chip is left in the tube (~1 min)
-	#~~Centrifuge for 5 min~~ at 80-100 x g ~~(RT~~).	+	# Place the vial in the hood and clean it with 70% ethanol
-	#~~Resuspend pelleted~~ cells in ~~appropriate volume of culture medium~~ and ~~culture o/n~~ in ~~CO2~~ incubator.	+	# Immediately remove the contents of the vial and place into the cold media
-	#~~Optional~~: ~~Exchange medium next day.~~	+	# 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==

Christopher C Vanlang: Thawing cells moved to TissueCulture:Thawing cells

2012-05-25T06:08:14Z

Thawing cells moved to TissueCulture:Thawing cells

←Older revision

Revision as of 06:08, 25 May 2012

Christopher C Vanlang: New page: ==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 e...

2012-05-25T06:08:05Z

New page: ==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 e...

New page

==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
*cryo tubes
*DMSO, Tissue culture (TC)-tested (e.g. [http://www.sigmaaldrich.com Sigma], D2650)
*Cell culture medium (depends on cell line/cells)
*Trypsin-EDTA (e.g. from [http://www.paa.com PAA], L11-004) - for adeherent cells
*'''Freeze-down medium'''
**6-10% DMSO
**Cell culture medium (I recommend to use at least 20% FCS and you can use 100% fetal calf serum (FCS) instead of cell culture medium)
*Basic TC centriguge (0 - 2500 rpm)
*Freeze-down box (this could be simply made from a small styropore box and cotton or from two styropore racks from 15-ml centrifuge tubes )

==Freeze-down (suspension cells)==
#Centrifuge at 80-100 x g for 4-5 min at RT.
#Suck out the medium.
#Resuspend 1-5 x 10*6 cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
#Transfer cryo-vials with cells into freeze-down box and store at -80 gr.C.
*Leave over-night at -80 gr.C.
*You can store cells at -80 gr.C for up to 6 months.
*Transfer cells into liquid nitrogen a day later for longer storage (1-20 years or so).

==Freeze-down (adherent cells)==
#Wash cells with PBS.
#Add thin layer of Trypsin-EDTA on cells (approx. 1ml for 90 mm Petri dish)
#Observe cells under microscope until these are rounded.
#Add serum-containing medium and resuspend cells. Transfer into centrifuge tubes.
#Centrifuge at 1000-1200 rpm for 4-5 min at RT.
#Suck out the medium.
#Resuspend cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
#Transfer cells into freeze-down box and place at -80 gr.C. (Comments as for suspension cells).

==Thawing==
#Keep frozen cells on dry ice until ready for thawing.
#Swirl a vial with frozen cells in 37gr.C water bath and remove just while a sliver of ice remains.
#Spray tube with 70% ethanol for sterilization.
#Aspirate cells from cryo-tube into pre-half-filled pipette with warm culture medium.
#Fill centrifugation tube with culture medium: at least 10 ml total volume.
#Centrifuge for 5 min at 80-100 x g (RT).
#Resuspend pelleted cells in appropriate volume of culture medium and culture o/n in CO2 incubator.
#Optional: Exchange medium next day.

==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.

Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.

==Contact==
*Who has experience with this protocol?

or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].


[[Category:Mammalian cell culture]]
[[Category:In vitro]]
[[Category:Protocol]]