Complete ES Cell Medium for C57BL6/N ES Cells

• 1. Supplement KO-DMEM (Invitrogen catalog no. 10829-018) with the following reagents and condition it by L-cells (CRL-2647, ATCC):

  Reagent	Final concentration
  FBS (ES cell tested; Hyclone)	10%
  l-glutamine	2 mm
  Penicillin and streptomycin	50 µg/mL each

• 2. Prepare the ES cell medium by combining L-cell conditioned medium (Step 1) with KO-DMEM (Invitrogen 10829-018) and KO Serum Replacement (Invitrogen 10828-028) in a ratio of 50:35:15.

  • For example, to prepare 100 mL: To 50 mL of the media prepared in Step 1, add 35 mL KO-DMEM and 15 mL of KO Serum Replacement. Then supplement the resulting medium as described in Step 3.

• 3. Supplement the resulting media with the following reagents:

  Reagent	Final concentration
  l-glutamine	2 mm
  Nonessential amino acids	0.1 mm
  Sodium pyruvate	1 mm
  β-mercaptoethanol	0.1 mm
  Penicillin and streptomycin	50 µg/mL each
  Insulin	4 µg/mL
  LIF (Millipore)	200 U/mL

• © 2017 Cold Spring Harbor Laboratory Press