Dicty Stock Center Cell Storage Procedure

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[INDEX]

1. Harvest 1-2 x10⁸ cells (1 clearing SM plate or axenic culture at appropriate density) for best viability results. 2 SM plates or 5 SM/5 plates for spores.
2. 
   
3. Pre-cool foam storage container in -20°C freezer. Pre-cool 10, 1.5-ml screw-cap storage vials in an ice water tray.
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5. Pour 20 ml cold HL5 into sterile 100-ml beaker.
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7. With sterile spreader, scrape cells or fruiting bodies from plates and transfer to beaker with HL5.
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9. Pour the cells from the beaker into a 50-ml conical tube.
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11. Rinse the beaker with 10 ml HL5 to collect remaining cells and then pour that into the conical tube as well.
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13. Replace cap and shake tube.
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15. For axenic strains, just pipette desired amount into conical tube.
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17. Centrifuge cells at 200xg for 5 minutes. Centrifuge spores at 500xg for 5 minutes.
18. 
    
19. Pour off supernatant.
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21. Resuspend pellet with 10 ml HL5 by vortexing (final density 1-2 x 10⁷ cells per ml).
22. 
    
23. Add 0.55 ml DMSO while gently swirling tube.
24. 
    
25. Aliquot 1 ml of cell suspension into 10 storage vials.
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27. Replace caps, tighten well, then dry vials with paper towel before placing into pre-cooled foam container.
28. 
    
29. Place foam container in -80°C freezer. Keep container away from metal to slow down the cooling rate.
30. 
    
31. After 24 hours, vials can be transferred to liquid nitrogen.
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After the vials have been in liquid nitrogen for several days, test the viability by scraping some cells from a vial (kept on dry ice) with a hot loop* and streaking them out on an SM/5 plate prespread with Klebsiella aerogenes. To test the viability and purity of an axenic strain, an entire vial should be thawed rapidly in a 37°C water bath and placed on ice as soon as the cell suspension has thawed. Do not allow cells to get too warm.

Pipette 50 µl of cells onto a plate with 0.25 ml K.a. and into 1 ml of HL5 in a 24-well plate. The remaining content of the vial can be transferred into a flask or a 100 x 20 mm style petri dish with 10 ml HL5.

* Even sturdy loops will bend out of shape, heat-kill some cells, and can melt the edge of storage tubes. A sturdy tool that gives much better plating results and no tube damage is Craftsman Professional No. 0 x 4 inch microtech Phillips head screwdriver, Sears # 00941527000 Mfr. Model # 41527. Heat the tip of the screwdriver and then cool in sterile water before using to scrape frozen cells from tube. Gently use tip of screwdriver to plate cells as usual.

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Notes

• Dictyostelium culturing procedures should take place in a sterile tissue culture hood. Plated cultures can be managed without it - though contaminants will be present. Axenic cultures, however, require sterile technique.
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• To prevent tubes from exploding, it is important to have the rubber o-ring stay in the cap during storage in liquid nitrogen. To keep o-rings from falling out, tighten caps all the way before initially opening vial.
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• If a vial is thawed, it should not be refrozen.
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• Viability dramatically decreases if cells are not immediately pipetted from the storage tube after thawing.
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• Storing cells at higher than suggested density decreases survivability.
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• We found that 5% DMSO gives better viabilities than 7.5% or 10% for our tested strains.
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• Storage in HL5 rather than horse serum is preferable if cells should ever need to be shipped internationally. There does not appear to be a difference in cell survivability between these two media.
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S. Marsh. Last updated July 14, 2005

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