Generating DMSO Stocks for Cell Culture - Revision history

Elhabbal at 18:34, 1 June 2018

2018-06-01T18:34:57Z

← Older revision		Revision as of 18:34, 1 June 2018
Line 8:		Line 8:
	#Pick a low passage number of cells and grow 2-5 10cm dishes.		#Pick a low passage number of cells and grow 2-5 10cm dishes.
	#At near confluence wash cells twice with PBS -/- and trypsinize normally.		#At near confluence wash cells twice with PBS -/- and trypsinize normally.
	#Collect all the cells in a 15 mL falcon tube. Add media up to 15 ml. (To avoid potentially over-trypsinizing, you can add media to each plate immediately after the cells detach, then add media up ~~tot eh~~ 15 ml mark on the falcon tube.)		#Collect all the cells in a 15 mL falcon tube. Add media up to 15 ml. (To avoid potentially over-trypsinizing, you can add media to each plate immediately after the cells detach, then add media up to the 15 ml mark on the falcon tube.)
	#Centrifuge 5 min at 1500RPM to pellet cells.		#Centrifuge 5 min at 1500RPM to pellet cells.
	#Aspirate media.		#Aspirate media.

Elhabbal: clarified some calculations, removed the isopropanol step

2018-06-01T18:34:21Z

clarified some calculations, removed the isopropanol step

Davebrid at 15:48, 13 August 2009

2009-08-13T15:48:31Z

← Older revision		Revision as of 15:48, 13 August 2009
Line 2:		Line 2:
	*Cells in 10cm dishes, at 90-95% confluence.		*Cells in 10cm dishes, at 90-95% confluence.
	*Cryopreservation Container (Nalgene 5100-0001)		*Cryopreservation Container (Nalgene 5100-0001)
	*Cryopreservation Vials Corning #430487		*Cryopreservation Vials (Corning 430487)
	*Sterile DMSO		*Sterile DMSO

Davebrid at 15:44, 13 August 2009

2009-08-13T15:44:03Z

← Older revision		Revision as of 15:44, 13 August 2009
Line 18:		Line 18:
	#Place container with vials at -80 for 1-3 days.		#Place container with vials at -80 for 1-3 days.
	#Remove cells from container and place in liquid nitrogen storage.		#Remove cells from container and place in liquid nitrogen storage.

			[[Category: Cell Culture]]
			[[Category: Storage]]

Davebrid at 15:41, 13 August 2009

2009-08-13T15:41:37Z

← Older revision		Revision as of 15:41, 13 August 2009
Line 2:		Line 2:
	*Cells in 10cm dishes, at 90-95% confluence.		*Cells in 10cm dishes, at 90-95% confluence.
	*Cryopreservation Container (Nalgene 5100-0001)		*Cryopreservation Container (Nalgene 5100-0001)
			*Cryopreservation Vials Corning #430487
	*Sterile DMSO		*Sterile DMSO

	==Protocol==		==Protocol==
	#Pick a low passage number of cells and grow 2-5 10cm dishes.		#Pick a low passage number of cells and grow 2-5 10cm dishes.
	#At near confluence wash cells twice with PBS -/- and trypsinize normally		#At near confluence wash cells twice with PBS -/- and trypsinize normally.
	#Collect all the cells in a 15 mL falcon tube		#Collect all the cells in a 15 mL falcon tube.
	#Centrifuge 5 min at 1500RPM to pellet cells		#Centrifuge 5 min at 1500RPM to pellet cells.
	#Aspirate media		#Aspirate media.
	#Add media (1.8 mL per original plate)		#Add media (1.8 mL per original plate).
	#Add sterile DMSO to a final concentration of 10% (0.2 mL per original plate)		#Add sterile DMSO to a final concentration of 10% (0.2 mL per original plate).
	#Gently resuspend cells and aliquot 1 mL of suspension into cryopreservation vials		#Gently resuspend cells and aliquot 1 mL of suspension into cryopreservation vials.
			#Label vials with name, date, cell type and passage (if known).
			#Ensure isopropanol is added to the container to above the indicated line.
			#Place container with vials at -80 for 1-3 days.
			#Remove cells from container and place in liquid nitrogen storage.

Davebrid: Created page with '==Materials== Cells in 10cm dishes, at 90-95% confluence. Cryopreservation Container (Nalgene 5100-0001) *Sterile DMSO ==Protocol== #Pick a low passage number of cells and gro...'

2009-08-13T15:35:11Z

Created page with '==Materials== *Cells in 10cm dishes, at 90-95% confluence. *Cryopreservation Container (Nalgene 5100-0001) *Sterile DMSO ==Protocol== #Pick a low passage number of cells and gro...'

New page

==Materials==
*Cells in 10cm dishes, at 90-95% confluence.
*Cryopreservation Container (Nalgene 5100-0001)
*Sterile DMSO

==Protocol==
#Pick a low passage number of cells and grow 2-5 10cm dishes.
#At near confluence wash cells twice with PBS -/- and trypsinize normally
#Collect all the cells in a 15 mL falcon tube
#Centrifuge 5 min at 1500RPM to pellet cells
#Aspirate media
#Add media (1.8 mL per original plate)
#Add sterile DMSO to a final concentration of 10% (0.2 mL per original plate)
#Gently resuspend cells and aliquot 1 mL of suspension into cryopreservation vials

← Older revision		Revision as of 18:34, 1 June 2018
Line 7:		Line 7:
	==Protocol==		==Protocol==
	#Pick a low passage number of cells and grow 2-5 10cm dishes.		#Pick a low passage number of cells and grow 2-5 10cm dishes.
	#At near confluence wash cells twice with PBS -/- and trypsinize normally.		#At near confluence wash cells twice with PBS -/- and trypsinize normally.
	#Collect all the cells in a 15 mL falcon tube.		#Collect all the cells in a 15 mL falcon tube. Add media up to 15 ml. (To avoid potentially over-trypsinizing, you can add media to each plate immediately after the cells detach, then add media up tot eh 15 ml mark on the falcon tube.)
	#Centrifuge 5 min at 1500RPM to pellet cells.		#Centrifuge 5 min at 1500RPM to pellet cells.
	#Aspirate media.		#Aspirate media.
	#Add media (1.8 mL per original plate).		#Add media (1.8 mL per original plate, so if you started with 5 plates, the added media will be 5*1.8).
	#Add sterile DMSO to a final concentration of 10% (0.2 mL per original plate).		#Add sterile DMSO to a final concentration of 10% (0.2 mL per original plate, so if you started with 5 plates, the added DMSO will be 0.2*5).
	#Gently resuspend cells and aliquot 1 mL of suspension into cryopreservation vials.		#Gently resuspend cells and aliquot 1 mL of suspension into cryopreservation vials. (Resuspend with a pipette tip gently.)
	#Label vials with name, date, cell type and passage (if known).		#Label vials with name, date, cell type and passage (if known).
	~~#Ensure isopropanol is added to the container to above the indicated line.~~
	#Place container with vials at -80 for 1-3 days.		#Place container with vials at -80 for 1-3 days.
	#Remove cells from container and place in liquid nitrogen storage.		#Remove cells from container and place in liquid nitrogen storage.