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Below is the recommended cell culture conditions for our cell lines within the bank. To find the medium for a particular cell line, please start from the Cell Bank holdings page, and click on the medium number listed for that cell line.

For melanocyte culture methods see Materials and Methods. All cultures are incubated at 37°C with 10% v/v CO2. This will result in an approximate pH of 6.9 - 7.0 with RPMI 1640 and Ham's, or pH 7.4 with standard DMEM. It is not recommended to use 5% CO2 with either medium as this will likely be too alkaline. However, if there is no other option,  medium can be acidified with HCl for use with 5% CO2 - see protocol.

Important : We recommend you do not heat foetal bovine serum, nor use heat-inactivated serum, as this destroys its stimulatory activity for melanocytes ( Bennett, D.C. et al. Development 105, 379-385 (1989).)

Abbreviations for components: (details of sources etc in Materials listing)

2-ME - 2-mercaptoethanol

CT - cholera toxin. (Caution: toxic, handle with care and treat media with bleach before disposal.)

EDN1 - endothelin 1

FBS - foetal bovine serum

FGF2 - fibroblast growth factor 2

HS - horse serum

hSCF - human stem cell factor

IBMX - 3-isobutyl-1-methylxanthine

mSCF - mouse stem cell factor

NCS - newborn calf serum

PTU - phenylthiourea (also known as phenylthiocarbamide) (Caution: toxic.)

RPMI - RPMI 1640

TPA - 12-0-tetradecanoyl phorbol acetate. TPA is hazardous - see Materials page for how to handle.

For melanocyte culture methods, see Materials Methods

Medium listings

RPMI 1640 ("RPMI") is supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), glutamine (2 mM), and additional phenol red (7.5 µg/ml), for easier monitoring of pH. See Materials for more details if required.

DMEM is supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml) and glutamine (4 mM).

We do not recommend adding growth factors (such as TPA or CT) directly to the bottle of medium, instead when required, aliquot medium and add growth factors to this before using.

  1. RPMI + FBS (10%) + TPA (200 nM). [some mouse melanocyte lines including melan-a]

  2. RPMI + FBS (10%) + TPA (200 nM) + CT (200 pM). [primary mouse melanocytes and some mouse melanocyte lines]

  3. Ham's F10 + FBS (20%) + TPA (48 nM) + IBMX (0.1 mM) at 33o. [DMEL lines from S. Prince with TS SV40 large T]

  4. RPMI + FBS (10%) + TPA (200 nM) + CT (200 pM) + 2ME (100 µM). [some mouse melanocyte lines]

  5. RPMI + FBS (10%) + TPA (200 nM) + 2ME (100 µM). [some mouse melanocyte lines including melan-c]

  6. RPMI + FBS (10%) + TPA (200 nM) + 2ME (100 µM) + PTU (200 µM). [some mouse melanocyte lines with abnormally high pigmentation] Further information on all media at top of page. For melanocyte culture methods see Materials Methods.

  7. RPMI + FBS (10%) + TPA (200 nM) + CT (200 pM) + 2ME (100 µM) + L-tyrosine (2 mM). [p-mutant mouse melanocyte lines]

  8. RPMI + FBS (10%) + TPA (200 nM) + CT (200 pM) + hSCF (10 ng/ml) + EDN1 (10 nM). [normal and immortal human melanocytes, new (SGM) RGP melanoma lines]. 8a: Same medium except 20 nM TPA, not 200 nM [New VGP melanoma line SGM5]

  9. RPMI + FBS (10%) + FGF2 (40 pM) + mSCF (20 ng/ml). (Earlier passages in RPMI + FCS (10%) + TPA(2 nM) + FGF2 (40 pM) on XB2-feeders). [mouse melanoblast lines]

  10. RPMI + FBS (10%) + TPA (200 nM) + CT (200 pM) + 2ME (100 µM). [some mouse melanocyte lines]

  11. RPMI + NCS (10%) + TPA (2 nM). [neural crest-like mouse stem cell lines]

  12. RPMI + FBS (5%). [mouse melanoma lines] 

  13. RPMI + FBS (10%). [human metastatic & some primary melanoma lines]

  14. DMEM + FBS (10%). [all fibroblasts except STO; Rama rat myoepithelial cells and XB2 mouse keratinocytes]

  15. DMEM + FBS (10%) + 2ME (100 µM) on gelatin*. [P19 mouse EC cells]

  16. DMEM + FBS (10%) on gelatin*. [C2 mouse myoblasts]

  17. 1:1 of Ham's F10:DMEM + 15% HS + 3% chick embryo extract on gelatin*. [MM14 mouse myoblasts]

  18. DMEM + FBS (5%). [RT4 rat pluripotent neuroblastoma lines]

  19. DMEM + FBS (10%) + hydrocortisone (50 ng/ml) + insulin (50 ng/ml). [Rama rat mammary glandular epithelial and mammary tumour lines

* gelatin is disolved in water (100 µg/ml) and used to pre-treat dishes.

 

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