Bennett Laboratory

An alphabetical listing of commonly used materials in our procedures, with sources and information

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A B C D E F G H I J K L M N O P Q R S T U V W X Y Z

A

B

beta-mercaptoethanol: see Mercaptoethanol.

bFGF (basic fibroblast growth factor), see FGF2

Index

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

Index

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.

Index

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 (technical details available online for this product using Adobe Acrobat Reader)

G

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

Glutamine/GlutaMAX
We buy a 200 mM 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.

Index

H

hSCF: see SCF

Hygromycin B
We get this from Sigma. Catalogue number H3274

I

J

K

Index

L

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

O

Index

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, 2.5 g KCl, 14.4 g disodium hydrogen orthophosphate, and 2.5 g potassium dihydrogen orthophosphate. We pH this stock to 7.2 and autoclave it. To use, we would dilute this solution 10 x and add 7.5 µg/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 104 units/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 3 mg/ml in distilled water, and autoclave to sterilize. We get the powder from Sigma, #P5530. Used at 1 in 400 to give a final 7.5 µg/ml in EDTA/PBSA solution and to add an extra 7.5 µg/ml to RPMI 1640 medium.

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

Index

Q

R

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

Index

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.

Index

T

TPA (12-O-Tetradecanoylphorbol 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 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.

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, 0.125 mg/ml) for human melanocytes and most melanoma cells
1:1 (1/2 dilution, 0.25 mg/ml) for mouse melanocytes, keratinocytes and epithelial cells
Used undiluted (0.5 mg/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 0.25 mg/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 = 750/4 µl =188 µl. So 188 µl 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

U

V

W

X

Y

Z

Index

 
 

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