Comparative Physiology @ Guelph
Filtering Hagfish Thread Cells
- 15 mL falcon tubes
- Millipore filtration system (250 ml upper portion, hollow lower stem with space for metal insert, circular metal grid insert for stem)
- 53um nylon mesh cut to size (stored in 5M NaOH)
- Vacuum flask
- Scintillation vial with lid
- Kim wipe and elastic to create a cover for the scintillation vial
- Petri Dish
- Lyophilizing jar with lid
- 500ml 1M Sodium Citrate solution
- 1M Sodium Citrate solution in wash bottle
- Collect fresh exudate into a 15 ml falcon tube containing 1M Sodium Citrate solution (stabilization solution) chilled to 4°C.
- Allow thread skeins to settle to the bottom of the falcon tube (approx 2-4 hours).
- Decant supernatant containing mucin vesicles so that concentrated thread skeins remain. Discard supernatant containing the mucin vesicles or save for future use.
- Immediately re-suspend tread cells in stabilization solution and invert gently until no pellet remains at the bottom of the falcon tube.
- Repeat the settling, decanting and re-suspending 4-5 times.
- Add DTT in the last re-suspension to make it 10 mM. Invert the flask multiple times. Decant the supernatant as soon as the thread skeins settle and replace it with fresh stabilization buffer.
- Once supernatant appears free from mucin, prepare filtering apparatus. Rinse all components with ddH2O. Remove mesh filter from 5M NaOH and rinse well with ddH2O then with stabilization solution in to waste. Allow the filter to sit in fresh stabilization solution in a Petri dish for several minutes.
- Assemble the filter apparatus ensuring there is no air between the mesh and the metal grid so that the stabilization solution flows through smoothly. Rinse the apparatus thoroughly with stabilization solution. This will ensure the thread skeins do not unravel when they come in contact with any remaining water.
- Pour the suspended thread cells into the filtration system. Rinse thoroughly, swirling the flask, and adding about 10 ml of stabilization solution from a wash bottle at a time. Rinse from the wash bottle, swirl and allow the solution to drip through, and repeat several times. Rinse for approximately 5 minutes, ensuring the threads do not dry out too much.
- Disassemble the filtration apparatus, and carefully rinse the thread skeins from the mesh into a scintillation vial with the wash bottle.
- Store the vial on an angle in the fridge so that the skeins fall into a corner of the vial. A small lid works well to ensure the vial does not fall over.
Preparation for lyophilization
The goal is to prepare the threads for lyophilization by replacing the salt with water. As thread skeins unravel easily in water, this must be done gradually and gently to preserve as much of the coiled threads as possible. Cold solutions may help. It is inevitable that some of the threads will unravel, and these are best avoided as much as possible when replacing the solutions. If some threads become attached to the pipette tip, do not attempt to pull them out, simply wrap the threads around the lip of the bottle and continue disturbing them as little as possible.
- Prepare dilutions of the stabilization solution (75%, 50%, 25%) and ddH2O in 50 ml falcon tubes and allow tochill to 4°C.
- Using the P5000 and the P1000 with the tip cut off, remove as much of the stabilization solution as possible.VERY gently, add approximately 10-15 ml of 75% stabilization solution and turn the vial gently to encourage mixing.
- Repeat the replacement with 50% stabilization solution, and twice more with 25%.
- Extremely cautiously, try a replacement with pure water. Do not feel the need to remove all of the water, but repeat this washing with pure water 4-5 times. It is important to get all of the salt out of the vial.
- Place vial and glass lyophilizing jar in -80°C freezer for at least 20 minutes.
- Prepare vacuum
- Turn freezer on, wait until it gets to -60°C
- Ensure all the valves (LHS) to be hooked up are closed
- Turn on vacuum, should go down from approximately 1000 atm to 15 atm
- Quickly remove sample from freezer, replace the cap with a kimwipe secured with an elastic band, and place in glass lyophilisis jar
- Attach jar to black top, and attach to the machine. Open the valve to the jar slowly. Watch the black top flatten under the pressure
- Leave on overnight, checking once or twice that the pressure has retuned to <20 atm after attaching the jar
- Once the lyophilization is complete, release the vacuum to the jar, and remove the jar and the sample. Shut down the vacuum system.
- Replace the kimwipe on the scintillation vial with a proper lid, and store in the regular freezer in a Tupperware containing desiccant.