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Melanoma stem cell media

 
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

[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

 

Components

HESCM4

[Cfinal]

V=250 ml

V=100 ml

HESCM

70%

175 ml

70 ml

HES

30%

75 ml

30 ml

  

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