Target Discovery and Validation Reviews and Protocols

230 Vijayaraj, Söhl, and Magin
f. Human insulin (cat. no. 0030110 SA, Invitrogen) stock solution at 4 mg/mL.
Aliquot as 2 mL and store at –20°C.
g. Progesterone (cat. no. P6149, Sigma): dissolve 1 mg in 1 mL ethanol, add 49 mL
of medium (1:1), and filter via 0.1-µm filter. Aliquot as 1 mL and store at –20°C.
h. Putrescin (cat. no. P6024, Sigma), dissolve in 6.2 mL of medium (1:1).
i. Na-selenite (cat. no. S 5261, Sigma) 10 mM stock I solution (0.865g/50 mL
cell culture grade H 2 O). To obtain stock II (100 µM), take 1 mL of stock I solution
and then complete to 100 mL with medium (1:1). Filter sterile (0.1-µm filter)
aliquot as 2 mL and store at –20°C.
j. BMP-4 (cat. no. 314-BP, R&D Systems): take 23,30 µL of 25% HCl solution
and complete to 100 mL with cell culture grade H 2 O (4 mM), and then filter
sterile (0.1-µm filter). To each BMP-4 vial add 1 mL of this solution and 1 µL
of BSA fraction V. Aliquot as 100 µL and store at 80°C.
For 500 mL medium:
225 mL DMEM-F12, 225 mL neurobasal medium, 10 mL Supplement B27,
5 mL BSA fraction V, 500 µL APO-transferrin, 3.125 mL human insulin, 300 µL
progesterone, 50 mL putrescin, 300 µL Na-selenite, 910 µL glutamine, monothioglycerol,
500 µL BMP-4, and 1 U/mL of final medium LIF. Store complete
medium at 4°C and use for up to no more than 1 wk.
2. Gelatin solution: use Sigma G2500 swine skin type I gelatin. Prepare a 1% solution
in cell culture grade water and autoclave. Leave overnight and autoclave again
the next day and store at 4°C. Warm up before preparing 0.1% working dilution.
3. Trypsin: for 500 mL of trypsin solution, mix the following: 400 mL of cell culture
grade sterile phosphate-buffered saline (PBS), 100 mL of sterile EDTA (1.85 g/L),
and 5 mL of trypsin (cat. no. 25090, Invitrogen). Mix and store as 100-mL aliquots
at –20°C. Once unfrozen, keep aliquots at 4°C.
4. ES cell freezing medium: although the ES cells are cultured in the absence of
serum, in our experience it is advisable to freeze ES cells in the presence of serum
(see item 5). On thawing, the cells are washed twice in serum-free medium before
plating.
5. Serum-containing culture medium: Glasgow modification of Eagle’s medium
(BHK-21 medium) without tryptose phosphate broth with L-glutamine (cat. no.
041-01710, Invitrogen), 5% 100 mM sodium pyruvate (Invitrogen), 5%
Nonessential amino acids (100X, Invitrogen), 5% Fetal calf serum (FCS)
(Sigma), 15% Monothioglycerol (Sigma), 1:50,000 LIF, 1 U/mL medium.
For homemade LIF, compare individual batches to commercial sources in a
functional assay.
KSOM medium complemented with amino acids: 95 mM NaCl, 25 mM
NaHCO 3 , 2.5 mM KCl, 10 mM lactate, 0.35 mM KH 2 PO 4 , 0.2 mM pyruvate,
0.2 mM MgSO 4 , 0.2 mM glucose, 1.71 mM CaCl 2 (2H 2 O), 0.2 mM L-glutamine,
0.01 mM EDTA, 1 mg/mL BSA; phenol red; 100X nonessential amino acids
(1:50); 50X essential amino acids (1:100).