Separate protocol available for primary culture.
Suggested growth medium:-
RPMI 1640, bicarbonate-buffered for pH 7.0-7.1 in your incubator.
It is easiest to use standard RPMI 1640 + 10%
CO2. If you cannot use a 10% CO2 incubator, you can
adjust the pH using acid or by using bicarbonate-free medium and adding less bicarbonate than normal; and pre-equilibrate with 5% CO2
(protocol available for that). However 10% gives best results.
If pH of medium rises during storage, readjust pH by gassing from cylinder of 10% (or 5% etc if using that) or 100% CO2 before adding to cells - alkaline medium is quite toxic to pigmented melanocytes. If gassing with 100% CO2, it is important not to over-gas (stop at orange color of phenol red, not yellow).
Penicillin, 105 U/L
Streptomycin, 100 mg/L
Glutamax or glutamine, 2 mM
Extra phenol red, 7.5 µg/mla
10% foetal calf serum (not heated - this impairs its stimulatory activity)
TPA, 200 nMb,c
Cholera toxin, 200 pMc
endothelin 1, 10 nMc,d
human stem cell factor (SCF), 10 ng/mlc
(higher concentrations better still, but it's expensive!).
Change medium 2 x per week.
Note: If you use flasks, it is helpful to gas flask with right %CO2 from cylinder, before adding cell suspension for plating out (since diffusion through neck of flask is slow), to avoid high-pH shock before equilibration in incubator.
Time between passages varies between strains, from a few days to several weeks. After a number of passages, growth rate starts falling as culture senesces, eventually to zero. Neonatal/infant melanocytes grow more quickly and for longer than adult melanocytes; they can reach 20-30 passages, 50-70 doublings in the recommended medium.
aPhenol red: we add this to help with monitoring pH of medium; RPMI has only 5 µg/ml, which is hard to see, especially in the presence of serum. We use water-soluble phenol red; see Materials.
bHandling of TPA: TPA (tetradecanoyl phorbol acetate) is a potent tumour promoter - care! It is also light-sensitive and quite unstable. A concentrated stock can be made at 2 mM in pure ethanol, stored at -70°C. (Do not use DMSO, which is toxic to melanocytes.) A working stock at 40 µM (200x) is prepared in phosphate-buffered saline containing BSA (say 1 mg/ml) or 1% serum as carrier, otherwise TPA from dilute solutions can stick to glass or plastic. Aliquots are stored at -70°C indefinitely, or at 4°C in dark for up to 2 weeks. TPA is added to medium on the day of use. TPA solutions and media are inactivated with bleach before disposal.
cSee Materials for sources of supplements etc. We use PBS + BSA carrier (as in note b) to dilute growth factor stocks. Aliquotted peptide solutions are kept at -70°C, or in fridge for up to 2 weeks
dWe have not tested endothelin 1 on primary explant cultures. It is a growth factor for some fibroblasts too, so it may stimulate contaminating cell growth.