Selection of ES Cells
Protocol for Serum-containing Medium
Note: Prior to selection, it is strongly recommended that a kill curve be determined for each ES cell line in order to determine the exact drug concentration to use. The following selection regimes can be used as a guide:
- Neomycin (G418) in 129SVEV and 129SVJ cells: 350 µg/mL for two days, then 150 µg/mL for the rest of the selection. Total 5–7 days selection.
- Neomycin (G418) in C57BL6/J cells: 275 µg/mL for one day, 200 µg/mL for the second day, then 150 µg/mL for the rest of the selection. Total 6–8 days selection.
- Hygromycin B in 129SVEV and 129SVJ cells: 100 µg/mL for two days, then 75 µg/mL for one day, then 50 µg/mL for the rest of the selection. Total 7–10 days selection.
Materials and Reagents
- ES Cell Medium supplemented with either Neomycin (G418) or Hygromycin B
- ES Cell Qualified DPBS — Millipore cat. nos. BSS-1006-A, BSS-1006-B
- Incubator, 37°C/5% CO2
- Pipette
Protocol
- To select for transformants, add ES cell medium containing either Neomycin (G418) or Hygromycin B.
- After 48 hours, cell death should be apparent. Change the culture medium daily if there is excessive debris evident or if the medium is discoloring, otherwise every other day is sufficient. If the debris is adhering to the living cells, wash the cells gently with sterile DPBS before changing the medium, taking care not to dislodge the feeder cell layer. ES cells should be cultured for approximately 10 days following electroporation.
Protocol for Serum-free Medium
Note: In serum-free medium conditions, a lower concentration of selection antibiotic is recommended.
Materials and Reagents
- ESGRO Complete Clonal Grade Medium — Millipore cat. no. SF001-500
- ESGRO Complete G418 Antibiotic — Millipore cat. no. SF011
- ESGRO Complete Puromycin Antibiotic — Millipore cat. no. SF012
- ESGRO Complete Hygromycin B Antibiotic — Millipore cat. no. SF013
- ES Cell Qualified DPBS — Millipore cat. nos. BSS-1006-A, BSS-1006-B
- Incubator, 37°C/5% CO2
Protocol
- To select for transformants, add ESGRO Complete Clonal Grade Medium containing ESGRO Complete G418, Hygromycin B, or Puromycin.
- After 48 hours, cell death should be apparent. Change the culture media daily if there is excessive debris evident or if the media is discoloring, otherwise every second day is sufficient. If the debris is adhering to the living cells, wash the cells gently with sterile DPBS before changing the media. ES cells should be cultured for approximately 5 to 10 days following electroporation
Electroporated ES cells (R1) grown in the presence of PMEFs and selected with G418 after 5 days of culture (10X). |
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