Aurelia Polyp Regeneration Protocol



*The term "wash" defined as brief vortexing (~10 seconds for cell resuspension), then immediately centrifuge at 3000 rcf for 5 minutes under room temperature. Followed by removal of supernatent fluid, and addition of desired solution.


1. One plate (~200 polyps) of Aurelia polyps were transferred to a beaker containing artificial seawater (ASW) and straved for approximately 60 hours.


2. After starvation, use a pestle to gently grind ~100 polyps/tube in a 1.5ml tube with 300ul 0.01M glycylglycine Calcium free ASW.  After grinding of polyps add 700ul 0.01M glycylglycine Calcium free ASW, vortex and centrifuge at 3000rcf for 5 minutes.


3. Remove supernate then wash once with 1ml 0.01M glycylglycine Calcium free ASW. 


4. Remove supernate then add 300 μL of 0.01M glycylglycine Calcium free ASW, 100 μL of 0.05% Trypsin EDTA (TRP) (1/4 of the total volume), and 5.0μL of PenStrep (10,000 units/mL penicillin + 10,000 μg/mL streptomycin), 1/100 of the total volume, to each microcentrifuge tube.


5. Vortex tubes for 10 seconds then rotate at 20rpm and room temperature for 3 hours. Remove tubes and re-suspend cell solutions every 30 minutes.


6. Vortex Tryptic digests for 10 seconds and filter with .0020-inch pore mesh into 50mL falcon tubes. Hold the mesh material taut over the 50ML falcon tube then pour the cell solution onto the mesh.  Use 4ML of 0.01M glycylglycine Calcium free ASW to wash the tube and mesh to be sure as many cells as possible end up in the falcon tube.


7. Centrifuge tubes @3000rcf for 5 minutes and remove supernate.  Then wash cells twice with 1ml natural seawater (NSW).


8. Remove supernate following the second natural seawater wash and add 100ul natural seawater to resuspend each pellet. Then combine the 4 aliquots to one 1.5ml tube and add 4.0μL of PenStrep.


9. Plate each sample in two wells of a round bottom 96well plate, 200ul in each well.  Let plate sit overnight.


10. After overnight incubation, transfer 100ul from one well (cut 200ul filter tip back a little to avoid damaging aggregates) to a 4 well plate and add 500ul NSW and 6ul penstrep. 


11. Observe daily.


Protocol based on Black and Riley, 1985