Culture Protocols

You will need the following list of supplies and reagents:

Item Vendor Catalog No.
KNOCKOUT DMEM Invitrogen 10829-018
0.25% Trypsin-EDTA Invitrogen 25200-056
β-mercaptoethanol Sigma M7154
FBS (ES cell qualified) Stem Cell Technologies 6952
Gelatin Millipore G-2500
L-Glutamine, 200mM Invitrogen 25030-081
LIF(ESGRO) Millipore ESG1107
Penicillin G ICN 194537
Streptomycin sulfate Invitrogen 11860-038
D-PBS Invitrogen 10010-023

Media Preparation

M15 Growth Media

GPS Solution (Glutamine/Penicillin/Streptomycin)

100 X ß-mercaptoethanol (BME)

MEF Feeder Media

Freezing Media (10% DMSO)

ES Cell Maintenance and Culture

Preparation of feeder plates

  • Add the appropriate volume of 0.1% gelatin solution to the plates and leave at room temperature for 30–60 minutes (See below: Table 1)
  • Aspirate the gelatin solution
  • Immediately add the appropriate volume and concentration of mitotically inactive MEF feeder cells (treated with mitomycin C or irradiated) to the plates (See below: Table 1)
  • Size of TC Plate Volume of Gelatin No. of Feeder Cells per Plate Volume of Media per Plate
    24-well 0.5 ml 1x10^5
    6-well 1.5 ml 5x10^5
    60 mm 2.0 ml 7x10^5
    100 mm 3.0 ml 1x10^6

Thawing the vial

Each vial contains a minimum of 2x106 ES cells and may be thawed on a 60 mm feeder plate:

Note: ES cells should be passaged every 2–3 days, and media should be changed prior to turning yellow.

Passaging ES cells

ES cells may be split 1:3 to 1:10:

Preparing ES cells for microinjection

ES cell culture after 24 hours, at approximately 40% confluence

Note: ES cell colonies with bright edges and a smooth, three-dimensional appearance.

images of cells

Freezing ES cells