Materials and methods

Suggested references : Sviderskaya EV et al, J. Inv. Dermatol. 108, 30-34 (1997)

Late embryos (14 days to term) can be used, but newborn mice up to 3 days old (no hair) give better yields. The younger, the better - the most melanocytes are obtained from newborn animals. One newborn mouse gives about 12ml of culture.

Melanocyte growth medium [for primary and early-passage cells] is RPMI-1640 medium with FCS (10%) (NOT heated - important!), TPA (200 nM) and cholera toxin (200 pM). Note that both TPA and cholera toxin are hazardous. Incubation is at 37°C with 10% CO₂, pH 7.0-7.1. This low pH is important for pigmented normal melanocytes, especially in early passages when they get very pigmented, as melanin synthesis becomes too rapid (toxic) at high pH. If you cannot use a 10% CO₂ incubator, you can adjust the bicarbonate level with acid (separate protocol available), but growth is less good that way. If you have repeated trouble with fibroblasts, try 5% FCS in place of 10%.

1 Prepare cultures of feeder cells (mitomycin C-treated XB2 keratinocytes) 1-3 days (preferably 1 day) earlier, in RPMI-1640 medium with 5% FCS.

2 Kill the animal(s), e.g. by decapitation.

3 Sterilise the skin of postnatal animals (not embryos) by immersing in 70% ethanol for 5-10 seconds (depending on age - shorter time for younger skin which is thinner; too long will damage the skin). All subsequent procedures are sterile.

4 Transfer to PBSA (Dulbecco's phosphate-buffered saline lacking Ca ++ and Mg ++ ), to wash off the ethanol.

5 Dissect off trunk skin in one piece, avoiding muscle (pinkish). Keep wet.

6 Collect skin(s) in PBSA and remove any bits of muscle using fine forceps and a dissecting microscope.

7 Transfer to concentrated trypsin solution, 5 mg/ml in PBSA (about 5 ml per skin).

8 Incubate at 37°C for about 1 hr for newborn skin. Embryonic skin is best trypsinized on ice until it will split: 15 min to 3 hr for 14-18 day embryos.

9 Transfer to PBSA, say 20 ml, in a large culture dish.

10 [The critical bit!] Split off epidermis (thin, silvery-white layer) under dissection microscope using 2 pairs of fine ("watchmaker's") forceps. It should come off very easily, otherwise trypsinize for longer. Use only epidermal tissue that splits off without any force - or you will get fibroblast contamination. Remove any obvious pieces of dermis (whitish), but don't scrape; melanocytes are loosely attached at this stage (so don't over-trypsinise either). This step generally takes some trial and error to learn.

11 Wash epidermis in clean PBSA.

12 Transfer to trypsin-EDTA solution as used for subculture (PBSA with trypsin, 250 µg/ml, and EDTA, 200 µg/ml), about 100 µl/skin, on a culture dish.

13 Chop quite finely with a curved scalpel.

14 Add 5 ml growth medium (lacking TPA and cholera toxin), with 5 µg/ml soybean trypsin inhibitor (yes, despite the serum), and pipette vigorously, 3-5 times, with a 5-ml pipette or a 2.5-ml Combitip.

15 Make up to about 12 ml with more growth medium and pipette 1-2 more times. Note: if feeder cells were plated only 1 day ago, it is beneficial to use the conditioned medium off the feeders for steps 14-15, instead of fresh medium (and work quickly to avoid drying-out of feeders).

16 Add TPA (200 nM) and cholera toxin (200 pM) and plate on to the feeder cells.

17 Change medium initially twice a week, but reduce frequency if cells become very sparse through loss of feeders. Unknown conditioning /autocrine factors seem important at this stage. Add new TPA twice a week when not changing medium, as it is unstable. Unpigmented melanoblasts are present initially, but groups of pigmented melanocytes should appear (readily identified by bright-field microscopy) from about 1-2 weeks. Skin keratinocytes also grow in the first week, but then differentiate and die.

Further procedures for early subcultures and establishing a line are here .

Suggested references: Sviderskaya EV et al, J. Inv. Dermatol. 108, 30-34 (1997) and Godwin, L. et al  Curr. Protoc. Cell Biol. 63:1.8.1-1.8.20

Primary culture is described above. The cells can be subcultured when approaching confluency or - if they are growing in isolated colonies - when the colonies get large (say 1000 cells or more each). This may be 3 weeks or more after primary culture. It is advisable to subculture cells at least every 2-3 weeks in any case, as mould spores can accumulate on outsides of dishes and eventually get inside [1].

Subculture by method described for immortal melanocytes , except plate on to fresh feeders, either at about 3-5x10⁴melanocytes/ml or at 1:1 [2], until immortal cells take over. This can be recognised because once the cells have senesced (around 4-6 weeks of culture - they get large, flat and well-pigmented), many cells fail to reattach at each subculture and only a small fraction even of surviving melanocytes grow and form colonies. The overall growth rate can fall to zero or below for some weeks or several passages, but do not discard. We think around 1 in 10⁶ normal mouse melanocytes spontaneously becomes immortal. When immortal cells appear, they form one or more healthy, growing colonies of small, less-pigmented cells, overgrow the static diploid cells, and virtually all survive and proliferate after subculture. If any melanocytes are proliferating (as opposed to surviving) by 3 months after primary culture, they are almost certainly immortal.

In general, after immortalization, feeder cells are no longer needed, and the cholera toxin can also be omitted. However feeder cells are needed again if cloning the immortal cells. Cloning is often useful to ensure homogeneity.

Hints:

[1] Watch for mould colonies on walls or shelves of incubator, and wipe away with 70% ethanol; in any case swab outsides of all long-term cultures and their incubator trays once a week with 70% ethanol.)

[2] Some early subcultures may have to be at 1:1 or thereabouts, when melanocytes are sparse. We suspect trypsin may then not be sufficiently diluted, as cell growth seems better on adding soybean trypsin inhibitor to resuspension medium. We use full amount needed to neutralise the trypsin. No need to remove it before plating cells. 1 mg/ml inhibitor stock in PBS is stable at 4 ° C for at least 2 months.

[3] FCS can be used at 5%, as higher concentrations often lead to appearance and overgrowth of fibroblast-like cells; but 10% gives faster growth of melanocytes and can be used once they are immortal and cloned.

[4] If fibroblasts do appear , they can be killed selectively by treatment with geneticin (G418) at 100-150 µ M for 3-4 days (no more). Melanocytes are very resistant to G418. Can be repeated after an interval. Physical scraping is sometimes useful to remove contaminating cells.

[5] Diploid melanocytes are even more sensitive to high pH than immortal ones. To help ensure cells are not exposed to alkaline medium during handling, we add extra phenol red indicator dye to the RPMI-1640 medium. RPMI-1640 contains 5 µg/ml phenol red (hard to see); MEM has 10 µg/ml and DMEM has 15 µg/ml. We add an extra 7.5 µg/ml to RPMI-1640. (We use water-soluble phenol red (sodium salt); can make stock at 3 mg/ml (400x); filter-sterilise; stable at room temperature).

See Sviderskaya et al (3) for most recent methods. For culture of our existing melanocyte lines, see Cell Bank Holdings  for specific medium recommended for each different line.

For sources and more information about materials, see Materials page.

CULTURE MEDIUM : RPMI 1640, gassed with 10% CO₂, pH 7.0-7.1. This low pH is essential only for pigmented (not albino) melanocytes. If pH of stored medium rises noticeably (red or pink colour), re-gas from cylinder of 10% CO₂ before use.

We strongly recommend use of a 10% CO₂ incubator for growing pigmented melanocytes. This should also be used for any cells grown in DMEM medium, as DMEM is buffered for 10% CO₂. If this is impossible and you can only use 5% CO₂ (or other concentrations of CO₂), you can EITHER start with bicarbonate-free medium; the [bicarbonate] to be added can be determined by a simple pH titration (4); OR add HCl and equilibrate pH by gassing from cylinder - other protocol available. However results are less good than with 10% CO₂.

Medium supplements:

penicillin, 100 000 U/L streptomycin sulphate, 100 mg/L glutamine 2 mM Foetal calf serum, 10%. ( Important: do not heat the serum, nor buy heat-inactivated serum, as this destroys its stimulatory activity for melanocytes [ref. 2].) TPA, 200 nM For some lines: cholera toxin, 200 pM We also add extra phenol red (water-soluble form, add 7.5 µg/ml from 3 mg/ml stock) to help monitor pH.

Hazard warning: TPA (tetradecanoyl phorbol acetate) is a potent tumour promoter - care with handling! It is also light-sensitive and quite unstable. A concentrated stock can be made at 2 mM in pure ethanol, stored at -70°C. Not DMSO: do not use DMSO as solvent. It is highly toxic to melanocytes. A working stock at 40 mM is prepared in phosphate-buffered saline containing 1 mg/ml BSA 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 the dark for up to 2 weeks. TPA is added to medium on the day of use. TPA solutions and media are inactivated with chlorine bleach before disposal.

2-Mercaptoethanol (ME): This is required for melan-c cells, melan-si-1 cells, and some others - see other web pages or publications. Worth trying, for a new cell line of your own. We think it inactivates something toxic in serum. ME stock (1 M) in water is stable only for 2-3 weeks, but culture medium + serum once treated with ME (0.1 mM) is ok for at least 2 months. Avoid rubber-lined caps which can cause a precipitate with concentrated ME. Add ME to medium a day (or at least 3-4 hr) before use to allow reaction.

ME (1 M) = 4.65 ml sterile water + 0.35 ml pure ME.

Alternative medium for albino melanocytes: Dulbecco's (DMEM) with 10% CO₂ (pH 7.4), with penicillin, streptomycin, pyruvate, glutamine (the 4 mM normal for DMEM is ok), 1% nonessential amino acids, mercaptoethanol (for melan-c and other cells that need it), FCS and TPA as above. Not suitable for pigmented melanocytes as pH 7.4 is high enough to be toxic for these. (DMEM with 5% CO₂ is even worse for melanocytes and at pH ~7.7 is not optimal even for fibroblasts etc, even though quite widely used).

PROCEDURES

Feeding: Medium is changed 2x weekly. If culture very sparse (under plating densities stated below) and fresh medium not needed after 3-4 days, add extra TPA as it is unstable.

Subculture: - For a growing culture, rinse in Dulbecco's phosphate-buffered saline lacking CaCl₂ and MgCl₂ (PBSA), and then in PBSA containing EDTA (200 µg/ml) and trypsin ( 250 µg/ml : less than for other cell types). OR, for a slowly-growing or quiescent culture, rinse 2x in EDTA solution and 1x in trypsin-EDTA. (The second method is best for melan-b at all times). Leave a little trypsin-EDTA on dish (e.g. 0.75ml for a 9-cm dish); incubate in (preferably) air at 37°C. - For a healthy single-cell suspension, do not pipette until the cells have not only detached from the dish but partly separated from each other. Check by microscope at intervals. Cells should dissociate sufficiently within about 4-6 min from a fast-growing culture, 8 min for more slowly-growing cells, 10-15 min for quiescent/confluent melanocytes. Best results are obtained with a growing culture - longer exposure to trypsin can cause some cell death. - Melan-a cells can be replated at 1.5x10⁴/ml every 5-7 days and should grow at least 20-fold; other lines generally at 3-5x 10⁴cells/ml whenever nearly confluent.

NOTE: Passage level: All these lines grow progressively faster with continued subculture. Melan-a cells, at least, become less pigmented. Especially if differentiated or "normal" cultures are important to you, it is recommended to freeze stocks immediately and use only passage numbers up to about 30.

References:

1 Bennett DC et al, Int. J. Cancer 39 , 414-8 (1987)

2 Bennett DC et al, Development 105, 379-85 (1989)

3 Sviderskaya EV et al, J. Inv. Dermatol. 108 , 30-34 (1997)

4 Bennett DC et al, J. Cell Science 77, 167-83 (1985)

This information is provided because it is sometimes difficult to freeze these cells. We have no evidence whether our basic method is any better for melanocytes than any other; if you have a different method, it may be fine. We only know that the "special precautions" (below) do help.

(1) Our basic method for cryopreservation (from M. Vogt)

Harvest cells as usual with trypsin and EDTA, resuspending in culture medium and serum and counting cell number.

Centrifuge (at about 1000 rpm for 7 min). Meanwhile prepare culture medium with serum (5% or 10%, as usual for that line) and DMSO, 7.5% v/v. Don't add neat DMSO to medium with cells in it. (Use glass pipette or micropipette tip for 100% DMSO - it dissolves something toxic out of other plastics such as plastic pipettes. So don't put neat DMSO in polystyrene plastic bottles or tubes either. Glass is ok.)

Remove supernatant and resuspend cells in the DMSO medium, at required density. We typically freeze 5-15 aliquots of 1-3x10⁶ cells each. Aliquot to freezing vials at 1 ml per vial.

Immediately place vials in insulated container at -70° C. Leave overnight (or up to a few days is ok without loss of viability), then transfer to liquid nitrogen freezer, ensuring their temperature does not rise above -40° C, which can kill cells. Use dry ice if necessary.

To recover stocks: vial is thawed quickly, e.g. in warm waterbath. Cells are resuspended from bottom of vial by gentle pipetting, transferred to a 20-ml centrifuge tube and diluted by gradually adding 20ml medium with serum. Important : dilute very slowly, dropwise . E.g. add first 1 ml with mixing over about 30 sec, second 1 ml over 10 sec, then remaining 18ml over 30 sec. (Note that freezing medium is highly hypertonic: over 4 x isotonic.) The DMSO needs time to diffuse out of the cells, or they burst. Centrifuge, resuspend in more medium and plate out at desired density (generally higher than normal).

(2) Special precautions for pigmented melanocytes, especially mouse :

We expect around 90-98% viability for most cell types frozen as above, but for very pigmented melanocytes (melan-a, melan-rs, melan-si-1, etc.) it can drop to 10% or even 1%. This can be improved by:

(1) Use of the tyrosinase inhibitor phenylthiourea (PTU), sold by Sigma as phenylthiocarbamide . Cells are grown with PTU (100-300 µM depending on level of pigmentation) for a few days, ideally a whole passage, before freezing; also after thawing for a few hours or days. We also add PTU at 200 µM to the solutions used for freezing and thawing the cells. This combination significantly increases recovery rates. The cells become depigmented. Pigmentation returns a few days after removing PTU. We dissolve PTU in ethanol at 10-100 mM and store aliquots at -20° C, or briefly at 4° C (The 100 mM solution precipitates when chilled, and should be warmed before use).

If cells are not completely depigmented, the following are also important:

(2) Ensure pH of culture medium is not too high (even for a few minutes) - do not use partially de-gassed medium, especially for the actual freezing medium.

(3) Work quickly, while cells are out of freezer but in the presence of DMSO , which is highly toxic to pigmented cells. (So are other cryopreservatives. We presume they permeabilize the melanosome membrane, with release of toxic melanin intermediates into the cytosol.)

Please note: this method is recommended only as a last resort, if it is impossible to set up a 10% CO2 incubator. The intracellular pH is affected more by CO₂/H₂CO₃ than by HCl, and better results are obtained that way. Required: - Hydrochloric acid, HCl, say 1M. Can assume it is sterile. - 1x culture medium (RPMI 1640). - Cylinder of compressed 5% CO₂ in air, with pressure reduction valve and outlet hose (plastic tubing), preferably with large 0.22 µm filter, to connect to sterile plugged pipette. Alternatively, cylinder of compressed air only, with hose etc. 1. Make up bottle of medium with glutamine, antibiotics etc (if not already included) but without serum (which would froth during gassing). We recommend adding extra phenol red to RPMI 1640 for easier monitoring of pH. Details under Cell Culture Media. 2. Add HCl at 10mM (final), e.g. 5 ml of 1M acid in 500 ml of medium. 3. pH initially becomes very low because medium is not yet in equilibrium with the atmosphere. This would be toxic to cells. Restore it to correct pH by gassing the medium thoroughly. In other words, bubble through it 5% CO₂ in air from the cylinder, to equilibrium (indicator colour stops changing). This can take a few minutes. Alernatively, carefully bubble plain compressed air until the phenol red indicator gives an orange colour (not yellow, not red). If neither option is available, it is possible to adjust the gas in the medium by leaving it overnight in the incubator with the screw top on but loose. But this warming will reduce its storage life. 4. NB, we don't know if 10 mM is right for all manufacturers' versions of RPMI. Incubators may vary a little too. If you are not getting an orange colour (pH 7.0-7.1) at equilibrium in the incubator, try adding a little more or less acid (by say 2-mM steps), until you are. 5. If pH of medium subsequently rises by de-gassing (CO₂ loss, medium goes pink) during storage, restore it by bubbling 5% CO₂ again before use. Even a few hours in alkaline medium is inhibitory for melanocytes. Or re-gas in incubator again. (Obviously you can't use a plain air cylinder to increase the CO₂ content, only to reduce it.) If your medium de-gases repeatedly before it is used up, try distributing it to smaller bottles.

XB2 is an immortal line of mouse keratinocytes that differentiated in culture from teratoma cells (J G Rheinwald & H Green, Cell 6 , 317-330 (1975)). Provided to us by JGR. XB2 cells were originally grown on feeder cells (3T3 fibroblasts) themselves, but we have selected a variant line which grows ok without fibroblasts (see Bennett et al, Int. J Cancer 39 , 414-8 (1987)).

(A) Growth of XB2 stock cells

For sources and more information about materials, see Materials page.

Medium : DMEM with 10% foetal calf serum, in 10% CO₂. Note: Standard DMEM medium is buffered for use with 10% CO₂, giving pH 7.4. With 5% CO₂ it is too alkaline (about pH 7.7). If you cannot get a 10% incubator, instead you can buy bicarbonate-free DMEM and make up the medium with about 25 mM bicarbonate instead of 45 mM, then gas with 5% CO₂ before use. On XB2 cultures, change medium 3- 4 days after plating. Subculture when cells virtually confluent but not yet at saturation density. Usually about 5-6 days.

Subculture procedure for a 9-cm dish: XB2 keratinocytes attach very firmly and need initial rinses in EDTA and a long time in trypsin to harvest them. (Solutions take a while to get underneath cells.) Also ensure that the cells are still growing, not at saturation density, when they get even harder to detach.

1) 2 washes (5 ml each) in Dulbecco's PBSA (PBS without Ca or Mg) containing EDTA (200  µg/ml).

2) 1 wash in the same EDTA solution with trypsin (250 µg/ml) (less trypsin than for fibroblasts, to allow for long exposure). Remove all but 0.75 ml of trypsin-EDTA.

3) Incubate at 37 ° C (preferably in air, not CO₂) until cells are completely detached, as judged by tilting dish. If a very small number of cells are still attached after a long time (say 15-20 min), stop incubation anyway. However the cells that take longest to detach seem to be the stem cells (small, flat, round, epithelioid, healthy-looking), so ensure nearly all cells are harvested . It may improve the culture to discard the quickest-detaching fraction every few subcultures, once familiar with the cells' appearance and the time taken to detach. The more-differentiated cells have a scrappy appearance and tend to fragment.

4) Resuspend in growth medium, count, replate at 3x10⁴/ml (10ml/9cm dish). With a healthy culture we get about 4-5x10⁵/ml after 6 days, i.e. doubling time about 1 .2 days. It is worth counting the cells, as they don't do well at densities that are either too low or too high.

(B) Preparation of feeders

Plate about four to six 9-cm dishes of cells as above. When they are ready to subculture, but still growing , remove medium and add 5 ml fresh medium containing 8 µg/ml mitomycin C*. Incubate for 3 to 3.5 hr (timing is important). Wash in DMEM and incubate in fresh DMEM+FCS without mitomycin, for 10 min, to elute any remaining drug. Then subculture as usual. Either replate at about 3x10⁴/ml for immediate use, or freeze aliquots of about 10⁶ cells in liquid nitrogen, and plate at about 5x10⁴/ml on thawing. Check viability of each batch on thawing (i.e. floating versus attached cells - none of them should divide); and adjust plating density for that batch if necessary.

Plate melanocytes on top within next 3 days, ideally after 1 day. Feeders gradually die off as melanocytes grow up. For preparative studies of immortal melanocytes that are feeder-dependent, like melan-si, these can be plated for 1 passage without feeders.

* Mitomycin C :- we get it from Sigma. Dissolved at 500µg/ml in water. Protect from light - wrap vials in foil during use. Can store in fridge for up to about 1 month only or indefinitely at -70C or less. However if freezing it, freeze rapidly to avoid precipitation, e.g. drop vials into liquid nitrogen. (We freeze it.)

This method is for normal human skin.

For sources and more information about materials, see Materials page.

Suggested basic growth medium :- RPMI 1640, bicarbonate-buffered for pH 7.0-7.1 in your incubator, and supplemented witrh FCS and several growth factors - details of supplements in Culture and Passaging protocol.

Explant Procedure :-

1 Provide nearly-full 20-ml tube of culture medium (with glutamine/Glutamax and antibiotics but serum and growth factors not needed), on ice, for collection of skin. (Neonatal or infant skin gives much better-proliferating cultures than adult: circumcisions a good source. Neonatal melanocytes are often unpigmented for first few passages.)

2 Soak tissue in 10ml of same medium with 10x normal [penicillin & streptomycin] for 10 min. All procedures sterile from here. Remove blood & blood clots (serum inhibits trypsin). Can trim to remove hypodermis (fatty layer).

3 Leave overnight ("16 hr") in 10 ml of ice-cold concentrated trypsin solution, 1.5 mg/ml in PBSA (calcium- & magnesium-free Dulbecco's phosphate-buffered saline, pH 7.2).

4 Transfer to 9-cm dish containing PBSA. Peel epidermis (thin, whitish) off dermis with sterile forceps. Transfer epidermis to universal.

5 Pipette well in medium + 10% FCS to disrupt tissue.

6 Make approximate cell count with haemocytometer. Make cell suspension to 5-10x10⁴cells/ml in complete growth medium. (around 1 ml per 5cm 2 growth area). Plate on dishes or flasks. If flasks, it is helpful to gas flask with right % CO₂ from cylinder, before adding cells (since diffusion through neck of flask is slow), to avoid high-pH shock before equilibration. Incubate.

Note: for a small sample it can increase yield and growth rate to plate on to mitomycin-treated feeder cells from an immortal line, XB2 mouse keratinocytes. Not essential for human melanocytes where SCF is used. See other protocol.

7 Change medium 2x weekly. Melanocytes should be apparent after 48 hrs, increasing to become predominant or only population.

8 If colonies of contaminating fibroblasts appear, treat with G418 (geneticin) at 75-150 µ M for 3-4 days. Repeat if necessary, but allow recovery for a few days between treatments.

9 Subculture (passage) when approaching confluence (cell yield =around 1.5 -2 x10⁵/ml). See Culture and Passaging . Avoid allowing normal cells to get confluent and quiescent - makes them harder to detach and reduces overall lifespan.

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.

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).

Supplements:- Penicillin, 10⁵U/L Streptomycin, 100 mg/L Glutamax or glutamine, 2 mM Extra phenol red, 7.5µg/ml a 10% foetal calf serum (not heated - this impairs its stimulatory activity) TPA, 200 nM b,c Cholera toxin, 200 pM c endothelin 1, 10nM c,d human stem cell factor (SCF), 10 ng/ml (higher concentrations better still, but it's expensive!).

Change medium 2 x per week.

Subculture procedure

• Subculture (passage) when approaching confluence (cell yield =around 1.5 -2x10⁵/ml). Wash gently 1x in PBSA, then 1x in PBSA containing EDTA (200µg/ml) and trypsin (125µg/ml: less than for other cell types).
• Remove trypsin immediately; add a small fresh amount of same solution to plate (e.g. 0.75 ml for a 10-ml plate).
• Incubate, preferably in air, until cells completely detached, as seen by gently tilting dish (4-10 min); do not attempt to detach them by pipetting or tapping - tends to kill them by breaking their processes .
• Resuspend in growth medium, count and replate at 5x10⁴cells/ml or more. If diluting less than 1:4, add some soybean trypsin inhibitor (use full amount to neutralise trypsin remaining on dish), or else centrifuge to remove trypsin. See also Materials .

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.

Notes:

a Phenol 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 .

b Handling 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 2mM in pure ethanol, stored at -70 ° C. (Do not use DMSO, which is toxic to melanocytes.) A working stock at 40µM (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 ind efinitely, 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.

c See 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

d We 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.

Hoechst 33258/ bisbenzimide stain for PPLO

Pigmented cells give false positives with some PPLO tests, owing to nonspecific binding of nucleic acids etc by melanin. However this test is ok for melanocytes.

A simple stain. Available as bisbenzimide from Sigma.

Cells are plated on dishes or coverslips (not flasks).

Fix after at least 2-3 days' growth but preferably not yet confluent. PPLO can be washed away at subculture, so more sensitive after a few days.

Fix/ permeabilize with fresh methanol-acetic acid, 3:1 and rinse 2 x in ethanol. Air-dry. (Can store at this stage).

Bisbenzimide stock solution can be made at 20-500µg/ml and stored in frozen aliquots if you snap-freeze in liquid nitrogen. Don't freeze slowly, or it precipitates and will not redissolve. Always handle dye in subdued light.

Use-solution of stain is at 0.2 µg/ml in PBS. No higher, otherwise mitochondria stain too. Dilute solution can be kept in refrigerator, but for no more than a few days, wrapped in foil.

Cover cells with stain solution; leave about 15 min (not critical).

Remove solution and rinse briefly 2x in distilled water. To view, either use water-immersion objective (in water), or mount with coverslip in UV-transparent mountant. View with fluorescence microscope, epi-illumination, and filters that transmit blue light, at high magnification. 20x objective or more.

This is a DNA stain. Nuclei should be bright blue.

Negative : bright, sharp, blue nuclei and cytoplasm totally invisible.

Positive : any kind of blue speckling in cytoplasm: some cells or all cells.

B

β-mercaptoethanol : see Mercaptoethanol.

bFGF (basic fibroblast growth factor), see FGF2

C

Cholera toxin We get this from Sigma . Catalogue number C8052

Note: it is becoming difficult in some areas to obtain cholera toxin. If necessary, it is possible to substitute other cAMP agonists , of which we would suggest the stable MSH analogue, 4-Nle, 7-D-Phe-alpha-MSH for mouse melanocytes and for human melanocytes from dark skin. However human melanocytes from pale skin may lack functional MSH receptors. Another commonly used cAMP agonist is IBMX (but we have no experience with it).

D

DMEM (Dulbecco's modification of Eagle's medium) We get the version containing Glutamax, from Gibco/Invitrogen . Catalogue no. 41966-021

DMSO (dimethyl sulphoxide) We get this from Sigma . Catalogue no. D8418

E

EDTA We buy this from Sigma as a 1 x solution in PBSA, to which we add 7.5 µg/ml of phenol red . Stored at room tempterature. Catalog no. E8008.

Endothelin 1 (EDN1) We get this from Bachem. Catalogue number 6995.

F

FCS (Foetal calf serum, also called Foetal bovine serum or FBS) We get this from Gibco/Invitrogen. Catalogue number 10270-106

FGF2 (Fibroblast growth factor 2, also called bFGF) We get this from R & D Systems Europe . Catalogue number 133-FB-025 

G

G418 (Geneticin) We get this from Gibco/Invitrogen . Catalogue number 11811

Glutamine/GlutaMAX We buy a 200mM stock solution from Gibco/Invitrogen , #25030. Used at 1 in 100, final 2 mM in RPMI 1640 medium, or 1 in 50 (4 mM) in DMEM. Aliquots are stored frozen. Or in some cases we use GlutaMAX, a useful alternative that is more stable.

H

hSCF: see SCF

Hygromycin B We get this from Sigma . Catalogue number H3274

M

Mercaptoethanol (= 2-mercaptoethanol or beta-mercaptoethanol) We get this from Sigma . Catalogue number M6250

Mitomycin C We get this from Sigma . Catalogue number M4287

mSCF : see SCF

MSH (melanocyte-stimulating hormone). We use the synthetic analogue 4-Nle, 7-D-Phe-alpha-MSH, which is stable in the presence of serum. Active at 1 nM for induction of differentiation; also as a growth factor (to substitute for cholera toxin) for mouse melanocytes, and for human melanocytes so long as they have active MSH receptors (MC1R). We get this from Sigma . Catalogue no. M8764.

N

NBCS ( Newborn Calf Serum) We get this from Gibco/Invitrogen . Catalogue number 16010-159

P

PBSA (Dulbecco's phosphate buffered saline, Solution A): (lacking CaCl₂ and MgCl₂ We get this from Gibco/Invitrogen . Catalogue number 14190.

We used to make our own PBSA as a 10 x stock solution. 1 litre contains 100g NaCl, 2.5g KCl, 14.4g disodium hydrogen orthophosphate, and 2.5g potassium dihydrogen orthophosphate. We pH this stock to 7.2 and autoclave it. To use, we would dilute this solution 10 x and add 7.5µg/ml of phenol red before adjusting pH once more and autoclaving. The pH of the solution sometimes alters during autoclaving and may need to be adjusted afterwards with sterile HCl or NaOH.

Penicillin/Streptomycin We buy a 100 x stock solution from Sigma , #P4333, containing 10⁴units/ml penicillin and 10 mg/ml streptomycin sulphate. Dilute 1 in 100 in medium, giving a final 100 U/ml penicillin and 100 µg/ml streptomycin sulphate.

Phenol Red (water-soluble) We make a 400 x stock solution with 3mg/ml in distilled water, and autoclave to sterilize. We get the powder from Sigma , # P5530 . Used at 1 in 400 to give a final 7.5µg/ml in EDTA/PBSA solution and to add an extra 7.5µg/ml to RPMI 1640 medium.

PTU ( Phenylthiourea, also called phenylthiocarbamide) We get this from Sigma . Catalogue number P7629

R

RPMI 1640 medium We get this from Gibco/Invitrogen . Catalogue number 318700025

S

hSCF ( human Stem Cell Factor) We get this from Invitrogen . Catalogue number PHC2111 (smaller sizes also available).

mSCF ( murine Stem Cell Factor) We get this from Invitrogen . Catalog number PMC2115 or depending on size.

T

TPA (12-O-Tetradecanoyl phorbol 13-acetate) Also called PMA, phorbol 12-myristate 13-acetate. FW 616.8 We get ours from Sigma . Catalogue number P8139

Hazard warning: TPA is a potent tumor promoter - care with handling! It is also light-sensitive and quite unstable. We make a concentrated stock at 2 mM in pure ethanol, stored at -70°C. Not DMSO: do not use DMSO as solvent. It is highly toxic to melanocytes. A working stock at 40 µM is also prepared, in phosphate-buffered saline containing 1mg/ml BSA 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 the dark for up to 2 weeks. TPA is added to medium on the day of use. TPA solutions and media are inactivated with chlorine bleach before disposal.

Trypsin We buy trypsin stock solution from Gibco/Invitrogen at 0.5 mg/ml in PBSA with EDTA, catalog # 2530054. Store frozen aliquots.

Trypsin-EDTA: We prepare this by mixing trypsin stock with EDTA solution in PBSA: 1:3 (1/4 dilution of trypsin, 0.125mg/ml) for human melanocytes and most melanoma cells 1:1 (1/2 dilution, 0.25mg/ml) for mouse melanocytes, keratinocytes and epithelial cells Used undiluted (0.5mg/ml) for fibroblasts

*Note* : commercial trypsin-EDTA mixtures are usually at the "fibroblast" concentration, and will need dilution in EDTA solution for melanocytes and keratinocytes, to avoid trypsin damaging cells.

Trypsin inhibitor (Soybean) We make up a stock solution of 1 mg/ml trypsin inhibitor in 1 x PBSA . Store small aliquots frozen. We use this at V/4, where V = the volume of 0.25mg/ml trypsin/ EDTA remaining on the culture dish to detach cells. E.g., by our protocol, a 90 mm dish will have 0.75 ml trypsin/ EDTA remaining. Therefore V/4 = 750/4µl =188 µl. So 188µl of trypsin inhibitor solution are added to halt the action of trypsin. LIkewise add V/8 to trypsin solution that is at 0.125 mg/ml.

We get ours from Sigma . Catalogue number T9128