Freezing of A1 cells: Difference between revisions

Latest revision as of 14:20, 8 June 2015

in one 175 cm2 flask

aspirate the old media from the cells
wash the cells 1 times with 5 ml APBS
remove the APBS
add 5 ml TE
wait till the cells are detached
stop the TE with HS – media
centrifuge at 1000 rpm, 3 min at 4ºC
aspirate the supernatant
dissolve the pellet into 1 ml freezing media
transfer the cells into the cryo tube and label the tube with the cell name, passage number and date
store the tube for one day at –80ºC
store the vial in liquid N2

@@ Line 1: / Line 1: @@
 ''in one 175 cm2 flask''
-Materials:
+== Material ==
--	APBS (100 ml PBS + 25 ml diwater)
+* APBS (100 ml PBS + 25 ml diwater)
--	TE in APBS (36 ml APBS + 4 ml TE)
+* TE in APBS (36 ml APBS + 4 ml TE)
--	1x 15 ml FALCON tubes
+* 1x 15 ml FALCON tubes
--	Pipettes
+* Pipettes
--	1 cryo tube
+* 1 cryo tube
--	freezing media
+* freezing media
-Procedure:
+== Procedure ==
+# aspirate the old media from the cells
--	aspirate the old media from the cells
+# wash the cells 1 times with 5 ml APBS
--	wash the cells 1 times with 5 ml APBS
+# remove the APBS
--	remove the APBS
+# add 5 ml TE
--	add 5 ml TE
+# wait till the cells are detached
--	wait till the cells are detached
+# stop the TE with HS – media
--	stop the TE with HS – media
+# centrifuge at 1000 rpm, 3 min at 4ºC
--	centrifuge at 1000 rpm, 3 min at 4ºC
+# aspirate the supernatant
--	aspirate the supernatant
+# dissolve the pellet into 1 ml freezing media
--	dissolve the pellet into 1 ml freezing media
+# transfer the cells into the cryo tube and label the tube with the cell name, passage number and date
--	transfer the cells into the cryo tube and label the tube with the cell name, passage number and date
+# store the tube for one day at –80ºC
--	store the tube for one day at –80ºC
+# store the vial in liquid N2
--	store the vial in liquid N2