Melanoma stem cell media

The Wistar Institute

3601 Spruce Street

Room 472

Philadelphia, PA 19104

1. Culture mouse embryonic fibroblasts (MEF) derived from E13-14 of CF-1 mouse.

2. Expose these cells to hES medium for 24 hours to produce condition medium (hESCM).

2. To prepare 1 liter of melanoma stem cells medium (hESCM4), mix hES and hESCM at the reation 30:70.

4. this medium can be used with or without adding fresh bFGF.

Components	HES
Components	[Cfinal]	V=250 ml	V=100 ml
DMEM/F-12 (Invitrogen Cat. No. 11330-032)	80%	200 ml	80 ml
KnockOut™ SR (Invitrogen Cat. No. 10828-028)	20%	50 ml	20 ml
L-glutamine (Invitrogen Cat. No. 21051-024; 0.146 g/10 ml DPBS, add 7 μl β-mercaptoethanol prior using)	100mM (β-mercaptoethanol 0.1mM)	1.25 ml	1 ml
MEM Non-Essential Amino Acids Solution 10 mM (100X), (Invitrogen Cat. No. 11140-050)	1%	2.5 ml	1 ml
bFGF (2 μg/ml)	4 ng/ml	0.5 ml	0.2 ml

Sterilized by using 0.22 μm CA filter

Cryo-preservative Medium for Melanoma Stem Cells (2X)
KnockOut™ SR (Invitrogen Cat. No. 10828-028)	30%
DMSO (Sigma Cat. No. D2650)	10%
HES	10%
HESCM4	50%

To freeze Melanoma Stem Cells: 1V of 2X Cryo-preservative medium + 1V of hESCM4

Meenhard Herlyn, D.V.M., D.Sc.