Day

Wash eggs well with 70% (v/v) ethanol.

Open the eggs by cutting a hole in the shell. Pluck embryos out with forceps, and place in a sterile 10-cm Petri dish.

3. Cut off and discard the head and limbs, and remove and discard the viscera.

4. Mince the torso into small fragments with a sterile razor blade.

5. Collect minced embryos using a wide-mouth 10-mL pipet. Place in a sterile 250mL Erlenmeyer flask with 10 mL of 1X Trypsin/EDTA for every four embryos.

6. Place flask on rotator, and gently swirl for 12-15 min at room temperature. You should see small clumps disappear, but you should not wait until they all disappear or the cells will start to die.

7. Take the flask off the rotator, and allow large clumps to settle. Transfer supernatant (i.e., leave clumps behind) to a sterile 50-mL plastic centrifuge tube.

8. Add an equal volume of fetal calf serum (FCS) to inhibit trypsin. Mix gently, and let stand for approx 5 min again to allow any big clumps to settle. Decant supernatant to a new tube.

9. Spin at low speed (approx 1000-2000g) for 5 min at room temperature to pellet cells.

10. Discard supernatant, and resuspend pellet in 25 mL of FCS. Count cell number to obtain an approximate idea of the number of cells/mL.

11. Pellet cells again (5 min at low speed as above).

12. Discard supernatant and resuspend cell pellet in 20 mL of CEF media (DMEM + 10% v/v FCS + 2% v/v CS + 1X pen/strep).

13. Plate on 10-cm tissue-culture dishes (see Note 5) in CEF media using a range of concentrations: e.g., 107 cells/dish, 3 x 106 cells/dish, 106 cells/dish, and 3 x 105 cells/dish. Incubate at 37°C in an 5% CO2 incubator.

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