Plating PMEF Feeder Cells
EmbryoMax Primary Mouse Embryo Fibroblasts (PMEF) feeder cells from Millipore are supplied as frozen vials containing 5–6 x 106 cells per vial at passage 3 (2–3 population doublings per passage). It is recommended that PMEF feeder cells be plated one day prior to plating ES cells, which guarantees approximately 95% confluence of the PMEF cells. If ES cells are plated earlier than one day after PMEF plating, there may be some small gaps in the feeder layer. Although plating ES cells when gaps are present may not have any detrimental effects on the ES cells, it is not recommended.
Materials and Reagents
- 15 mL tubes
- Centrifuge
- EmbryoMax cryopreserved PMEF feeder cells
- Gelatin coated tissue culture plates or flasks
- Incubator, 37°C/5% CO2
- PMEF Feeder Cell Medium
- Pipette
- Water Bath, 37°C
Protocol
- Prior to thawing PMEF feeder cells, coat plates or flasks with gelatin solution.
- Thaw PMEF vial(s) quickly in a 37°C water bath and transfer to a 15 mL tube (already containing 10 mL of warm PMEF Feeder Cell Medium). Gently invert the tube to distribute, and centrifuge at 300 xg for 4–5 minutes.
- Remove supernatant and resuspend the cell pellet in warm PMEF Feeder Cell Medium.
- Remove the Gelatin solution from plates or flasks, and aliquot the PMEF feeder cell suspension at the densities recommended in the table provided.
- Incubate the PMEF Feeder cells at 37°C with 5% CO2. Use images provided as a guide for an estimate of correct PMEF density and appearance. Gelatinized plates containing PMEF may typically be used for 12–14 days before discarding.
PMEF feeder cells at the | PMEF feeder cells at too low density | PMEF feeder cells at too high density |
Recommended PMEF Feeder Cell Suspension Densities
| Dish Size | Volume (mL.)/flask or well | Growth Area (cm2) | No. of feeder cells/flask or well |
| 75 cm2 flash | 12 | 75 | 3.75 x 106 |
| 25 cm2 flask | 6 | 251.5 | 1.25 x 106 |
| 100 mm plate | 1- | 56 | 2/8 x 106 |
| 60 mm plate | 5 | 21 | 1.0 x 106 |
| 6-well plate | 4 | 9.5 | 4.75 x 105 |
| 12-well plate | 2 | 4 | 2.0 x 105 |
| 24-well plate | 1 | 2 | 1.0 x 105 |
| 96-well plate | 0.1 | 0.32 | 1.5 x 104 |
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