Protocol for Airbrushing tissue from coral

1. Materials
   • Iwata airbrush
   • Compressed air source
   • Tissue homogenizer
   • 10 % Bleach
   • DI water
   • Isopropanol wipes
   • ice-cold 1X PBS (pH = 7.4)
   • forceps

2. Protocol

   1. Remove coral fragment from freezer
   2. Use forceps to place coral fragment in labeled ziplock bag (quart size)
   3. Airbrush with 1X PBS (pH = 7.4)
      • Take care to remove tissue from entire surface area, including deep tissue
      • Attempt to keep total blastate volume between 15-8 mL to fit into one falcon tube (i.e., use PBS through airbrush efficiently!).
   4. Transfer tissue slurry from the bag to a sterile 15mL falcon tube and rinse sides of bag with 1x PBS, adding rinsate to falcon tube.
   5. Record total volume of tissue slurry and store on ice until homogenization
   6. Homogenize slurry for 30 sec in falcon tube using the Bio-Gen PRO200 Homogenizer . If blastate is in multiple falcon tubes, homogenize each and pour back and forth between tubes to ensure even mixing.
   7. Between each sample, clean the forceps and the homogenizer in a 10% bleach solution for 30 seconds, DI wash #1 for 30 seconds, DI wash #2 for 30 seconds, then wipe down with an isopropanol wipe.

Coral Airbrushing Video

Store homogenized tissues on ice until ready to aliquot for downstream assays *****

1. Aliquots for downstream assays (see flowchart below)

   • Label six 1.5-mL microcentrifgue tubes for each sample.
     • See labelling scheme below: A-Host, A-Sym-1, A-Sym-2, B-Host, B-Sym, C)

• Add 1 mL homogenized tissue blastate to three of the 1.5-mL tubes (Tubes: A-Sym-1, B-Sym, C).
  • Set aside C tube. This is the Holobiont fraction for protein, carbohydrate, and lipid assays - Centrifuge tubes A-Sym-1 and B-Sym 1.5-mL tubes at 13,000g for 3 min.
• From A-Sym-1 tube:
  • Pipet off supernatant (~1mL) and transfer to A-Host tube. This fraction is for host protein, carbohydrate, and lipid assays
  • Resuspend pellet in 1mL 1xPBS. Vortex thoroughly and pipet up and down to fully dissolve pellet.
  • Transfer 200uL to the A-Sym-2 tube. this fraction is for symbiont cell counts
  • Keep the remaining 800uL in the A-Sym-1 tube. this fraction is for symbiont protein, carbohydrate, and lipid assays
• From B-Sym tube:
  • Pipet off supernatant (~1mL) and transfer to B-Host tube. This is extra host fraction
  • Store this pellet as is in the B-Sym tube. This is the chlorophyll assay tube
• Freeze the remainder of the homogenate for Ash-free dry weight (5 mL)