Cell dissociation protocol

STG cell culture

General considerations:

1) Keep everything you need labeled with your name, dates, and the word STERILE with big red letters as well as all kinds of credible life threats. You don’t want anybody to touch this stuff without your knowledge. There is a 1000% guarantee that will contaminate your materials, both because they don’t know and because they don’t care as much as you do.

2) Use gloves and sterilize them by splashing a little bit of 70% ethanol on them frequently. Ethanol is a good bactericide and works best at this concentration. There is a dogma that says that ethanol does not NOT sterilize as well at higher concentrations. I suspect that it does not improve anymore above 70% and thus you can save about 30% of ethanol.

3) Anything that can’t be autoclaved must be sterilized in 70% ethanol:
    - Your dissection tools (they could be sterilized in the autoclave really).
    - Plastic Petri dishes (those lined with Sylgard)
    - Anything else

4) Be careful not to add too much ethanol to your preparation as you dissect it because it will eventually kill it. A few small drops on your dissection tools are OK. More than that will require that you replace the saline more often with fresh sterile saline plus antibiotics.

5) Always have ready the following, aside from your sterilized dissection and culturing material:
- Chilled saline (~ 1 lt) with antibiotics. All the solutions you use should contain 0.1 mg/ml gentamicin (a broad spectrium bactericide).  The fungicide Fungizone may be necessary as well.
- 70% ethanol
- Zero Ca⁺⁺/zero Mg⁺⁺ saline sterile solution with 0.1 mg/ml gentamicin
- Chilled culture medium: 50% solution of L-15 Leibovitz medium + 50% Cancer supplement solution 
plus 0.1 mg/ml gentamicin + 2 mM glutamine.    
- Fire polished pipettes. These are made of glass electrode tubing pulled on a puller to make microelectrodes. Their tips are broken against a fine  sharpening stone or sandpaper and then firepolished under a low heat flame (alcohol Bunsen burners are good for this) to achieve the desired internal diameter of the pipette  (~50-100 microns or so; check under the microscope against the real cells to get a good idea of the diameter you need!). Make sure to have many ready and sterilized (autoclaved) since you never know how each will work or how many you will brake.

Procedure:

1) Anesthetize your crab in ice

2) Wipe the dorsal carapace with a tissue saturated with 70% ethanol

3) Dissect the STNS with sterilized tools into a previously sterilized (autoclaved) black dish

2) Dissect out ganglia and necessary nerves. Then transfer STG and other nerves and ganglia to a 35mm Sylgard-lined (sterile) Petri dish and pin them down. Use saline with antibiotics all the time !!

3a) Rinse the preparation once in sterile zero Ca⁺⁺/zero Mg⁺⁺ saline solution + antibiotics.

3b) If the cells are to be tracked over time later on after dissociation, this is the moment to label them with a dye, such as DiI.  How???  We are trying to find out.

4) Incubate at room temperature for 60 - 120 minutes in the proteolytic enzyme dispase at 2% (dissolved in zero Ca⁺⁺/zero Mg⁺⁺ saline with antibiotics). Activity of the enzyme may vary from batch to batch and over time, and tissue strength may also vary. Thus, see how well the duration of this incubation works.

5) Wash out the enzyme with chilled zero Ca⁺⁺/zero Mg⁺⁺ saline once or twice.

6) Replace solution with culture medium (50% Supplement solution + 50% Leibovitz L-15 medium) + antibiotics

7) Make sure the ganglion is well pinned to the Sylgard (sometimes a pin straight through the neuropile can be necessary if the nerves and sheath have been too digested by the enzymes)

8) Proceed to dissociate cells by suction into the firepolished pipettes. Suction is best applied by mouth using a long flexible tube that has some sort of holder that you can clamp with your teeth and that has a small filter (0.2 micron pore size diameter) as well as a piece of cotton stuck somewhere along its length to prevent you from contaminating the cells if your mouth gets too "watery". Also suck gently and avoid fluid going past the glass pipette into the plastic tubing to minimize contamination. When a cell has been dissociated from the ganglion, it has to be transferred to Nunclon 35 mm Petri dishes prefilled to a 1-2 mm depth with culture medium. More than one cell can go into a dish but not too many. If electrophysiology is to be performed, usually only one cell per dish can be used. A good yield is about 30-50%. So, expect 1 cell to survive every 2 or 3 cells that you plate. Also, try to transfer cells to the center of the Petri dish.

9) Once cells have been plated, the Petri dish should be covered and then left completely undisturbed for at least 1 hour to allow cells to adhere to the bottom. After that time they must be sealed with Parafilm (to avoid culture medium concentration changes due to evaporation) and then transferred (VERY gently) to an incubator.

Copyright: STG Lab 2006
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Last Modified:March 06, 2009