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Melanoblast media

MCDB 153

Order from Sigma Cat. No. M-7403


Stock Concentration: 1X


Preparation: Resuspend powder in ddH2O, add Sodium Bicarbonate, adjust pH, then filter-sterilize (done by Cell Center). Please read instruction from Sigma for details of preparation using this link. 
Final Concentration: 87%


Store at 4°C, light-protected (up to 3 weeks).

Use 87 ml / 100 ml total medium.

 

FETAL BOVINE SERUM (FBS)

Order from Cansera Cat. No. CS-C08-100.

Stock Concentration: 1X


Final Concentration: 2%


Preparation: Heat in manufacturer’s bottle at 56°C for 20 minutes
.

Store at 4°C (up to 3 weeks).

Use 2 ml / 100 ml total medium.

 

CHELATED FBS

Order Chelex 100 (chelating resins) from Sigma Cat. No. C-7901


25 g = $ 32.60


Preparation: Dissolve 15 g Chelex in 500 ml FBS, stir 1.5 hrs at 4°C
 Filter-sterilize with 0.2 μm filter


Stock Concentration: 1X


Final Concentration: 10%


Aliquot into 50 ml aliquots


Store at -20°C.

Use 10 ml / 100 ml total medium.

 

L-GLUTAMINE

Order from Mediatech Cat. No. MT 25-005-CI

Stock Concentration: 200 mM


Final Concentration: 2 mM


Preparation: Aliquot into 10 ml aliquots.


Store at -20°C.

Use 1 ml / 100 ml total medium.

 

CHOLERA TOXIN

Order from Sigma Cat. No. C-8052


500 µg = $52.00


Stock Concentration: 40 nM; 3.32 µg/ml


Final Concentration: 20 pM; 1.66 µg /L


Preparation: Dissolve 500 µg in 150 ml ddH2O and filter-sterilize (0.2 µm, non-absorbent filter).


Aliquot 140 ml into 14 ml aliquots and 10 ml into 250 µl aliquots.
Store at 4°C (up to 1 year).

Do not freeze!


Use 50 µl / 100 ml total medium.

 

bFGF (Recombinant Human bFGF)


Obtain from E. coli in concentration of 4 µg / µl or 222 µM in 10% glycerol in PBS. Or order from RDI Cat. RDI-118bx

1 mg = $1400


Stock Concentration: 0.14-0.57 µg/ml; 31-126 nM


Final Concentration: 1.1-4.5 ng/ml; 60-250 pM


Preparation: Dissolve 4 µg in 7 ml of 0.1% BSA in PBS and filter-sterilize (0.2 µm, non-absorbent filter). 
Aliquot into 500 µl aliquots.


Store at -20°C


Use 200 µl / 100 ml total medium.

 

ET3 (Recombinant Human, rat, pig, rabbit)

Order from Peninsula Cat. No. 6911 500 µg = $295.00

or from American Peptide Company Cat. No. 88-5-10, 1 mg = $275; 5 mg = $1100, 10 mg = $1975


Stock Concentration: 100 µM; 264 mg/ml


Final Concentration: 100 nM; 264 ng/ml


Preparation: Dissolve 500 µg in 1.89 ml of 0.1% BSA in PBS and filter-sterilize (0.2 µm, non-absorbent filter)
Aliquot 1 ml into 200 µl aliquots and rest in 100 µl aliquots


Store at -20°C.

Add 100 µl / 100 ml total medium.

 

SCF (Recombinant Human)

Order from R&D Systems Cat. 255-SC-050, 50 µg = $960.00

or from Leinco Technologies Inc. Cat. No. S109, 1 mg = $3200


Stock Concentration: 10 µg/ml


Final Concentration: 10 ng/ml


Preparation: Add 50 µg SCF to 5 ml of 0.1% BSA in PBS and filter-sterilize (0.2 µm, non-absorbent filter). 
Aliquot 3 ml into 500 µl aliquots and 2 ml into 100 μl aliquots


Store at -20°C.

Use 100 µl / 100 ml total medium.

 

 

For Preparation of Stock Solutions:

DULBECCO’s PHOSPHATE BUFFERED SALINE (DPBS) without Ca2+ , without Mg2+

Order from Cellgro Cat. No. MT 21-031-CM


Stock Concentration: 1x


Store at 4°C .

 

BOVINE ALBUMIN (BSA) Fraction V, 96-99%, cell culture tested


Order from Sigma Cat. No. A-9418


10 g = $45.70


Preparation: Dissolve 0.1 g of powder in 100 ml HBSS (without Ca2+ and Mg2+) and filter-sterilize (0.2 µm, non-absorbent filter).


Final Concentration: 0.1 %


Store at 4°C (up to 2 months).

 

Note: Before filtering stock solutions in 0.1 % BSA/PBS pre-wet the filter with enough 0.1% BSA/PBS! This is to avoid loss of the precious growth factors.

 

Components

Melanoblast

[Cfinal]

V = 512 ml

MCDB153

87%

87 ml

Fetal Bovine Serum (FBS)

2%

2 ml

Chelated FBS

10%

10 ml

L-glutamine

5 μg/ml

1 ml

Cholera toxin

15 μg/ml

50 μl

bFGF

0.5 ng/ml

200 μl

ET3

 

200 μl

SCF

1.68 mM

100 μl

Sterilized by using 0.22 μm filter

 

Optional supplements:

LEUKEMIA INHIBITORY FACTOR


Obtain from supernatant of SNL/STO cells


Stock concentration: 1X


Final Concentration: 10%


Preparation: After three days of culture, collect supernatant and filter-sterilize (0.2 mm, non-absorbent filter).


Store at -20° C
Or

Order from Gibco Cat. No. 18276-829


10x106 units = $1000


Use 10 ml/100 ml = 100 units/ml

 

GENTAMICIN SULFATE


Order from Cellgro Cat. No. MT 30-005-CR

Stock Concentration: 50 mg/ml


Final Concentration: 50 mg/ml


Store at room temperature

 

HEPARIN sodium salt (grade I-A: from porcine intestinal)

Order from Sigma Cat. No. H3149170 usp units/mg


Final Concentration: 1 ng/ml