Freezing of C2C12 cells: Difference between revisions

Latest revision as of 14:30, 8 June 2015

@@ Line 1: / Line 1: @@
-Materials:
+== Material ==
+* 15 ml FALCON tube
+* PBS w/o Ca2+ and Mg2+
+* TE in PBS
+* Freezing media (10% DMSO + 40% serum + 50% DMEM)
+* Pipettes and tips
+* Glas pipetts
+* cryo tube
+* ice
+* freezing box
--	15 ml FALCON tube
+== Procedure ==
--	PBS w/o Ca2+ and Mg2+
+# wash the cells with 5 ml PBS
--	TE in PBS
+# remove the PBS and add 2 ml TE
--	Freezing media (10% DMSO + 40% serum + 50% DMEM)
+# let it incubate for 3 – 5 min
--	Pipettes and tips
+# stop the TE with 5 ml HS – media and resuspend the cells
--	Glas pipetts
+# pipette the cell suspension back in the FALCON tube
--	cryo tube
+# centrifuge the cells (1000 xg, 3 min at 4 ºC)
--	ice
+# remove the old media
--	freezing box
+# resuspend the cells 1 ml freezing media
+# quickly pipette the liquid in the cryo tube
-procedure:
+# put the cells in the freezing box and store this for one day at –80 ºC
+# place the cells one day later into the liquid nitrogen
--	wash the cells with 5 ml PBS
--	remove the PBS and add 2 ml TE
--	let it incubate for 3 – 5 min
--	stop the TE with 5 ml HS – media and resuspend the cells
--	pipette the cell suspension back in the FALCON tube
--	centrifuge the cells (1000 xg, 3 min at 4 ºC)
--	remove the old media
--	resuspend the cells 1 ml freezing media
--	quickly pipette the liquid in the cryo tube
--	put the cells in the freezing box and store this for one day at –80 ºC
--	place the cells one day later into the liquid nitrogen