Marek: Freeze-down/Thaw - Revision history

Mcebe at 12:09, 17 September 2007

2007-09-17T12:09:24Z

←Older revision		Revision as of 12:09, 17 September 2007
Line 12:		Line 12:
	*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'''		*'''Freeze-down medium'''
-	**6% DMSO ~~(some people prefer 10%)~~	+	**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)		**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)
Line 23:		Line 23:
	#Transfer cryo-vials with cells into freeze-down box and store at -80 gr.C.		#Transfer cryo-vials with cells into freeze-down box and store at -80 gr.C.
	*Leave over-night at -80 gr.C.		*Leave over-night at -80 gr.C.
-	*You can store cells at -80 gr.C for 6~~-12~~ months.	+	*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).		*Transfer cells into liquid nitrogen a day later for longer storage (1-20 years or so).

Mcebe at 12:08, 17 September 2007

2007-09-17T12:08:08Z

←Older revision		Revision as of 12:08, 17 September 2007
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	#Suck out the medium.		#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).		#Resuspend 1-5 x 10*6 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.	+	#Transfer cryo-vials with cells into freeze-down box and store at -80 gr.C.
	*Leave over-night at -80 gr.C.		*Leave over-night at -80 gr.C.
	*You can store cells at -80 gr.C for 6-12 months.		*You can store cells at -80 gr.C for 6-12 months.

Mcebe at 12:06, 17 September 2007

2007-09-17T12:06:58Z

←Older revision		Revision as of 12:06, 17 September 2007
Line 11:		Line 11:
	*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	+	*'''Freeze-down medium'''
	**6% DMSO (some people prefer 10%)		**6% DMSO (some people prefer 10%)
	**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)		**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)
Line 18:		Line 18:

	==Freeze-down (suspension cells)==		==Freeze-down (suspension cells)==
-	#Centrifuge at ~~1000~~-~~1200 rpm~~ for 4-5 min at RT.	+	#Centrifuge at 80-100 x g for 4-5 min at RT.
	#Suck out the medium.		#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).		#Resuspend 1-5 x 10*6 cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).

Mcebe at 12:05, 17 September 2007

2007-09-17T12:05:52Z

←Older revision		Revision as of 12:05, 17 September 2007
Line 13:		Line 13:
	*Freeze-down medium		*Freeze-down medium
	**6% DMSO (some people prefer 10%)		**6% DMSO (some people prefer 10%)
-	**Cell culture medium (I recommend to use at least 20% FCS and you can use 90% fetal calf serum (FCS) instead of cell culture medium)	+	**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 box (this could be simply made from a small styropore box and cotton or from two styropore racks from 15-ml centrifuge tubes )
Line 20:		Line 20:
	#Centrifuge at 1000-1200 rpm for 4-5 min at RT.		#Centrifuge at 1000-1200 rpm for 4-5 min at RT.
	#Suck out the medium.		#Suck out the medium.
-	#Resuspend cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).	+	#Resuspend 1-5 x 10*6 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.		#Transfer cells into freeze-down box and place at -80 gr.C.
	*Leave over-night at -80 gr.C.		*Leave over-night at -80 gr.C.
Line 38:		Line 38:
	==Thawing==		==Thawing==
	#Keep frozen cells on dry ice until ready for thawing.		#Keep frozen cells on dry ice until ready for thawing.
-	#~~Warm-up~~ cells ~~by shaking/incubation~~ in 37gr.C water bath ~~until little~~ ice ~~cube visible~~.	+	#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 ~~to sterilize~~.	+	#Spray tube with 70% ethanol for sterilization.
	#Aspirate cells from cryo-tube into pre-half-filled pipette with warm culture medium.		#Aspirate cells from cryo-tube into pre-half-filled pipette with warm culture medium.
-	#~~Resuspend cell in~~ culture medium: 10 ml total volume.	+	#Fill centrifugation tube with culture medium: at least 10 ml total volume.
-	#Centrifuge for 5 min at ~~1000 rpm~~ (RT).	+	#Centrifuge for 5 min at 80-100 x g (RT).
-	#Resuspend pelleted cells in appropriate volume of culture medium.	+	#Resuspend pelleted cells in appropriate volume of culture medium and culture o/n in CO2 incubator.
		+	#Optional: Exchange medium next day.

	==Notes==		==Notes==

Mcebe at 12:01, 17 September 2007

2007-09-17T12:01:17Z

←Older revision		Revision as of 12:01, 17 September 2007
Line 13:		Line 13:
	*Freeze-down medium		*Freeze-down medium
	**6% DMSO (some people prefer 10%)		**6% DMSO (some people prefer 10%)
-	**Cell culture medium (you can use ~~100~~% fetal calf serum (FCS) instead of cell culture medium)	+	**Cell culture medium (I recommend to use at least 20% FCS and you can use 90% 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 box (this could be simply made from a small styropore box and cotton or from two styropore racks from 15-ml centrifuge tubes )

Mcebe: /* Thawing */

2007-06-12T14:29:25Z

Thawing

←Older revision		Revision as of 14:29, 12 June 2007
Line 37:		Line 37:

	==Thawing==		==Thawing==
		+	#Keep frozen cells on dry ice until ready for thawing.
		+	#Warm-up cells by shaking/incubation in 37gr.C water bath until little ice cube visible.
		+	#Spray tube with 70% ethanol to sterilize.
		+	#Aspirate cells from cryo-tube into pre-half-filled pipette with warm culture medium.
		+	#Resuspend cell in culture medium: 10 ml total volume.
		+	#Centrifuge for 5 min at 1000 rpm (RT).
		+	#Resuspend pelleted cells in appropriate volume of culture medium.

	==Notes==		==Notes==

Mcebe: /* Materials */

2007-05-18T11:28:26Z

Materials

←Older revision		Revision as of 11:28, 18 May 2007
Line 12:		Line 12:
	*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		*Freeze-down medium
-	**6% DMSO	+	**6% DMSO (some people prefer 10%)
	**Cell culture medium (you can use 100% fetal calf serum (FCS) instead of cell culture medium)		**Cell culture medium (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)

Mcebe: /* Freeze-down (adherent cells) */

2007-05-18T10:05:55Z

Freeze-down (adherent cells)

←Older revision		Revision as of 10:05, 18 May 2007
Line 34:		Line 34:
	#Suck out the medium.		#Suck out the medium.
	#Resuspend cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).		#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.	+	#Transfer cells into freeze-down box and place at -80 gr.C. (Comments as for suspension cells).
		+
		+	==Thawing==

	==Notes==		==Notes==

Mcebe: /* Freeze-down (suspension cells) */

2007-05-18T10:05:03Z

Freeze-down (suspension cells)

←Older revision		Revision as of 10:05, 18 May 2007
Line 25:		Line 25:
	*You can store cells at -80 gr.C for 6-12 months.		*You can store cells at -80 gr.C for 6-12 months.
	*Transfer cells into liquid nitrogen a day later for longer storage (1-20 years or so).		*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.

	==Notes==		==Notes==

Mcebe: /* Materials */

2007-05-18T10:02:04Z

Materials

←Older revision		Revision as of 10:02, 18 May 2007
Line 6:		Line 6:
	List reagents, supplies and equipment necessary to perform the protocol here.		List reagents, supplies and equipment necessary to perform the protocol here.

-	*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)		*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)