Feeder-free and Serum-free ES Cell Culture
Using ESCRO Complete System Protocol
Procotol
- Grow mouse ES cells to 60% confluence in serum-supplemented medium in a T25 flask with or without feeders.
- Change medium 24 hours prior to seeding into serum-free conditions.
- Pre-coat T25 flasks with Gelatin Solution.
- Wash cells once with DPBS. To dissociate cells, add 1 mL Accutase solution. Incubate at 37°C and allow cells to detach (5–10 minutes). Tap gently and add 5 mL of Basal Medium (Millipore cat. no. SF002-100), mix and spin at 1000 rpm.
Note: It is important not to use standard trypsin. “Balling” of ES cells can occur.
- Remove supernatant and resuspend pellet in 5 mL Clonal Grade Medium. Count cells.
- Plate 1x106 cells into a pre-coated T25 flask containing 10 mL pre-warmed Clonal Grade Medium.
- Observe cell growth over the next 2–3 days. Some residual feeders may remain stuck down, some cell death may be observed and some differentiation may be visible. However, ES cell colonies will continue to grow and may appear to be flatter than those on feeders with a distinct nuclear and cytoplasmic morphology.
- When mES cells are about 60% confluent, perform a 1 in 5 split of the T25 flask into another coated T25 flask containing Clonal Grade Medium.
Note: Do not let ES cells in serum-free media become over confluent as they will begin to differentiate.
- To passage the ES cells, remove the media and wash with DPBS buffer. Add 1 mL of Accutase solution per T25 flask. Note: Do not use standard trypsin.
- Return to 37°C incubator and allow cells to detach (5–10 minutes). Tap gently and add 5 mL Basal Medium; mix and spin at 1000 rpm.
- Remove supernatant. Resuspend pellet in 5 mL Clonal Grade Medium.
- Cell growth should be observed over the next 2–3 days. The ES cells should be passaged as in steps 9 and 10 an additional 2–3 times. After these passages no or few residual feeders should remain and the remaining ES cells should have only low levels of differentiation.
Product News & Releases
- Faster Flow Rates for Sterile Filtration - New Millex® syringe filters with Millipore Express PLUS (PES) membrane
- ReNcell® VM Neural Stem Cell Line Models Parkinson’s Disease - New human disease model shows function of PINK1 gene in neural degeneration
- Millipore Issues New Laboratory Filtration Product Guide
- Millipore Launches Progenitor Cell-Enriched Primary Human Epithelial Cell Cultures
Featured Products
- Fast-Trap Lentivirus Purification and Concentration Kit
- Fast-Trap Adenovirus Purification and Concentration Kit
- EasyCyte Plus Flow Cytometry System
- ECM Cell Culture Optimization Arrays
- ESGRO Complete
- GCTM-5 Antibody, clone GCTM-5
- Guava Viacount Assay
- MilliTrace Constitutive GFP Reporter
- MultiScreenHTS + and MultiScreen Vacuum Manifolds
- ENStem-A hES-derived Human Neural Progenitors
Pathways
