Preparation of High Serum for A1 cells: Difference between revisions

Latest revision as of 14:28, 8 June 2015

For 400 ml

@@ Line 1: / Line 1: @@
-For 400 ml
+== Material ==
+''For 400 ml''
+* FCS 				40 ml
+* Eagle’s MEM			252 ml
+* DdH2O				100 ml
+* Pen/ Strep			4 ml
+* Glutamin			4 ml
+* Insulin (stock: 1mg/ ml)	4 ml
-FCS 				40 ml
+== Procedure ==
-Eagle’s MEM			252 ml
+# grow at 25ºC in 2% CO2
-DdH2O				100 ml
+# if you want to grow without CO2, then substitute L-15 for the MEM
-Pen/ Strep			4 ml
-Glutamin			4 ml
-Insulin (stock: 1mg/ ml)	4 ml
--	grow at 25ºC in 2% CO2
+==== passage once per week 1:3 ====
--	if you want to grow without CO2, then substitute L-15 for the MEM
--	passage once per week 1:3
+# wash with APBS (100 ml PBS + 25 ml ddH2O)
+# trypsinize with TE
+# stop with media
+# centrifuge cells at 100 rpm 3 minutes
--	wash with APBS (100 ml PBS + 25 ml ddH2O)
+==== to make myotubes: ====
--	trypsinize with TE
--	stop with media
--	centrifuge cells at 100 rpm 3 minutes
-to make myotubes:
+# wash cells 1x with APBS
+# put cells in 0.5% FCSmedia
--	wash cells 1x with APBS
+# wait 3 days
--	put cells in 0.5% FCSmedia
--	wait 3 days