Electroporation of DAPI into Dictyostelium

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[MATERIALS] - [INDEX]

Procedure

1. Give fresh HL5 medium to all plates to be resuspended.
2. 
   
3. Resuspend wildtype cells from 2 or 3 plates and transfer to a sterile 50 ml conical tube.
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5. Pellet cells by spinning in tabletop centrifuge at 2,000 rpm for 5 minutes.
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7. Draw off HL5 medium and replace with COLD H50 buffer. Be sure to not disturb the cell pellet. Resuspend cells to wash and pellet as before.
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9. Draw off 1st H50 wash and replace with a 2nd 10ml aliquot. Resuspend cells to wash again and remove a small aliquot using a Pasteur pipette and bulb. Add a drop of resuspension to each side of grid on hemacytometer. Gently place cover glass over grid.
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11. Note remaining volume of resuspension and pellet as before.
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13. Count cells on grid and determine the total number of cells in tube.
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15. Draw off 2nd H50 wash and replace with an appropriate volume of H50 such that the final cell density is 5 x 10⁷ cells/ml.
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17. Thaw DAPI in DMSO stock solution. Dilute DAPI in 100 µl cell/H50 suspension such that final DAPI concentration is 1.5 µg/ml. Cell suspension and DAPI are mixed in a 0.5 ml microfuge tube. Use DAPI stock solution at a concentration such that the amount of DMSO added to reaction is minimized (use highest concentration of DAPI stock solution possible).
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19. Place cells/DAPI in a pre-chilled 0.1 mm cuvette; leave cuvette on ice for 3 minutes.
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21. Place cuvette in cold cuvette holder. Zap (pulse or electroporate) cells @ 0.85 kV and 25 µF by depressing two red buttons simultaneously until a beep is heard. Record time constants.
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23. Immediately following electroporation, add 1 ml of HL5 medium to cuvette. Leave cuvette at room temperature for 10 minutes.
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25. Use gel loading pipette tip to remove ~200 µl aliquots from cuvette (don't forget to remove cells between electrodes) and add to a coated coverslip.
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27. Allow cells to settle onto coverslip for 1hr before adding an additional 1 ml HL5 medium to well. Add medium to edge of well such that cells attached to coverslip are not disturbed. Allow cells to recover for additional 3 hrs.
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29. Observe cells on an inverted scope using oil immersion lens and DAPI filter. Neutral density filters may be needed to minimize glare caused by intense staining.
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Materials

• H-50 buffer
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  • 20mM Hepes
  • 50mM KCl
  • 10mM NaCl
  • 1mM Mg₂SO₄
  • 5 mMNaHCO₃
  • 1mM Na₂HPO₄
  
  Adjust pH to 7.0

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