Reagents and equipment

• Isolated ICMs from cynomolgus monkey blastocysts (Protocol 1)

• Feeder layers in 4-well dishes: seed mitomycin C-treated MEFs or STO cells (Chapter 2) onto gelatin-coated wells (Protocol 1, footnote c, Chapter 6) at a density of 1.5-2 x 104 cells/cm2

• CEM, or culture medium for cES cells: for 500 ml CEM, supplement 400 ml of a 1:1 mixture of DMEM/F12 (Cat. No. D6421, Sigma) with the following additives; 5 ml of 100 x non-essential amino acids (Cat. No. M7145, Sigma), 6.25 ml of 200 mM L-glutamine (Cat. No. G7513, Sigma), 4 ml of b-mercaptoethanol (final concentration 0.1 mM; Cat. No. 137-06862, WAKO), and 100 ml Knockout™ Serum Replacement (KnockOut™ SR; Cat. No. 10828-028, Invitrogen). Mix, and store at 4 °C for up to 2 weeks

• LIF- and bFGF-supplemented CEM: cES-cell medium supplemented with 4 ng/ml recombinant, human basic fibroblast growth factor (bFGF: Cat. No. 01-106, Upstate Biotechnologies) and 10 ng/ml recombinant, human leukaemia inhibitory factor (LIF; Cat. No. LIF1010, Chemicon International)a

• PBS, Ca2+- and Mg2+-free (Cat. No. D8537, Sigma)

• Dissociation solution I: to 59 ml PBS add 10 ml of 2.5% trypsin solution (Cat. No. 15090-046, Invitrogen), 10 ml of 10mg/ml collagenase IV (Cat. No.17104-019, Invitrogen) in PBS, 20 ml of KnockOut™ SR, and 1 ml of 100 mM CaCl2. Aliquots should be stored at-20 °C, and thawed only once

• Aspirator tube assembly (Protocol 1)

• Inverted phase-contrast microscope

• Stereodissecting microscope

• 27 Gauge hypodermic needle

• Stereodissecting microscope

• 27 Gauge hypodermic needle

• Drawn-out glass capillaries, inner diameter 200-300 mm (see Protocol 1)

0 0

Post a comment