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: 80%
Store at 4°C, light-protected (up to 3 weeks). Use 87 ml / 100 ml total medium
Order from Cansera Cat. No. CS-C08-100. Lot No. SF80508
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
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
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 |
80% |
87 ml |
|
Fetal Bovine Serum (FBS) |
20% |
2 ml |
|
Chelated FBS |
2% |
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
Usee 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 intestnal)
Order from Sigma Cat. No. H3149
170 usp units/mg
Final Concentration: 1 ng/ml