Melanoma Isolation Media
Tu2% is used for majority of melanoma cell lines.
|
Components |
Tu2% |
||
|
[Cfinal] |
V=511 ml |
V=102 ml |
|
|
MCDB153 |
80% |
400 ml |
80 ml |
|
Leibovitz’s L-15 |
20% |
100 ml |
20 ml |
|
Fetal Bovine Serum (FBS) |
2% |
10ml |
2 ml |
|
Insulin (Bovine) |
5 μg/ml |
0.5 ml |
0,1 ml |
|
CaCl2 |
1.68 mM |
0.42 ml |
0.084 ml |
Sterilized by using 0.22 μm filter
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: 80%
Store at 4°C, light-protected (up to 3 weeks).
Use 400 ml / 511 ml total medium.
LEIBOVITZ’S L-15
Order from Mediatech Cat. No. MT 10-045-CV
Stock Concentration: 1X
Final Concentration: 20%
Store at 4°C.
Use 100 ml / 511 ml total medium.
FETAL BOVINE SERUM (FBS)
Order from Cansera Cat. No. CS-C08-100
Stock Concentration: 1X
Final Concentration: 2%
Preparation: Heat in manufaturer's bottle at 56°C for 20 minutes.
Store at 4°C (up to 3 weeks).
Use 10 ml / 511 ml total medium.
INSULIN (BOVINE)
Order from Sigma Cat. No. I-5500
500 mg = $72.00
Stock Concentration: 5 mg/ml
Final Concentration: 5 µg/ml
Preparation: Dissolve 500 mg of insulin in 100 ml of sterile 10-4 M HCl in ddH<sub>2</sub>O (= 1 ml of 0.01 M HCl and 99 ml of sterile ddH2O). Aliquot into 2 ml aliquots.
Store at 4°C (up to 4 months).
Use 0.5 ml / 511 ml total medium.
CALCIUM CHLORIDE (CaCl 2)
Order from Sigma Cat. No. C-5670
100g = $30.30
Stock Solution: 2 M
Final Concentration: 1.68 mM
Preparation: Dissolve 44.4 g CaCl 2 in 200 ml ddH2O, filter-sterilize (0.2 µm filter). Aliquot into 1 ml aliquots.
Store at Room Temperature.
Use 420 µl / 511 ml total medium at room temperature and discard if any precipitation occurs.
Mel2% is used for establishing melanoma cell lines and culturing WM 3523/3523 and WM373
|
Components |
Mel2% |
|
|
[Cfinal] |
V=512 ml |
|
|
MCDB153 |
80% |
400 ml |
|
Leibovitz’s L-15 |
20% |
100 ml |
|
Fetal Bovine Serum (FBS) |
2% |
10 ml |
|
Insulin (Bovine) |
5 μg/ml |
0.5 ml |
|
Bovine Pituitary Extract (BPE) |
15 μg/ml |
0.6 ml |
|
Epidermal Growth Factor (EGF) |
5 ng/ml |
0.5 ml |
|
CaCl2 |
1.68 mM |
0.42 ml |
Sterilized by using 0.22 μm filter
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: 80%
Store at 4°C, light-protected (up to 3 weeks).
Use 400 ml / 512 ml total medium.
LEIBOVITZ’S L-15
Order from Mediatech Cat. No. MT 10-045-CV
Stock Concentration: 1X
Final Concentration: 20%
Store at 4°C.
Use 100 ml / 512 ml total medium.
FETAL BOVINE SERUM (FBS)
Order from Cansera Cat. No. CS-C08-100
Stock Concentration: 1X
Final Concentration: 2%
Preparation: Heat in manufaturer's bottle at 56o C for 20 minutes.
Store at 4°C (up to 3 weeks).
Use 10 ml / 512 ml total medium.
INSULIN (BOVINE)
Order from Sigma Cat. No. I-5500
500 mg = $72.00
Stock Concentration: 5 mg/ml
Final Concentration: 5 µg/ml
Preparation: Dissolve 500 mg of insulin in 100 ml of sterile 10-4 M HCl in ddH2O (= 1 ml of 0.01 M HCl and 99 ml of sterile ddH2O). Aliquot into 2 ml aliquots.
Store at 4°C (up to 4 months).
Use 0.5 ml / 512 ml total medium.
BOVINE PITUITARY EXTRACT (BPE)
Order from Clonetics Cat. No. CC-4009
Stock Concentration: 13 mg/ml
Final Concentration: 15 µg/ml
Preparation: Aliquot into 300 µl aliquots.
Store at 70° C.
Use 0.6 ml / 512 ml total medium.
EPIDERMAL GROWTH FACTOR (From mouse submaxillary glands)
Order from Sigma Cat. No. E-4127
Stock Concentration: 5 pg/ml
Final Concentration: 5 ng/ml
Preparation: Dilute stock in 0.1% BSA in HBSS. Aliquot into 500 µl aliquots.
Store at 20$de C (up to 3 months).
Use 0.5 ml / 512 ml total medium.
CALCIUM CHLORIDE (CaCl2)
Order from Sigma Cat. No. C-5670
100g = $30.30
Stock Solution: 2 M
Final Concentration: 1.68 mM
Preparation: Dissolve 44.4 g CaCl2 in 200 ml ddH2O, filter-sterilize (0.2 µm filter). Aliquot into 1 ml aliquots.
Store at Room Temperature
Use 420 µl / 512 ml total medium at room temperature and discard if any precipitation occurs
For Preparation of Stock Solutions:
HBSS without Ca2+ and without Mg2+
Order from Invitrogen Cat. No. 14175-079 Or from Cell Center
Stock Concentration: 1X
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)
Mel/TPA is sued for cell line WM1650
|
Components |
Final Concentration |
|
RPMI |
- |
|
FCS |
6% |
|
Cholera toxin |
10-12 M |
|
TPA |
10-7 M |
Culturing WM 1650
Please remember a few things about this line as it is an EXTREMELY slow grower and even more painful to defrost:
- Use a 1% gelatin coat for at least 10 minutes in the incubator before defrosting the cells onto your flasks.
- Use 1% gelatin coats each time you split the cells. We recommend defrosting these cells into either a T25, or 2 wells of a 6-well plate.
- Please be patient with this line and refresh media 2x/week. We would not recommend splitting the cells higher than a 1:2 ratio. Also, please try not to split cells until they are 90-95% confluent. They sustain themselves better when they have good cell-cell interaction.
Please seek the following Cell Lines through the ATCC: http://www.atcc.org/SpecialCollection/wisar.cfm
451Lu
1205Lu
WM35
WM39
WM115
WM266-4
WM278
WM793
WM1552C
