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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