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