Isolation of Single Fibers for Seahorse Assays

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This protocol is modified from https://www.agilent.com/cs/library/applications/5991-7148EN.pdf

Materials

  • Seahorse Plate (XF24) and Cartridge
  • Matrigel, slowly thawed and aliquoted into 100 uL aliquots. Stored at -20
  • KRBH Buffer, about 20 mL pre-warmed to 37C
  • Collagenase
  • DMEM with no Phenol (Invitrogen cat# [21063-029]), need about 20 mL
    • Prepare by adding 2% FBS (400 uL) and 200 uL PSG to a 50 mL conical tube
  • Assay Media - aCSF Media, about 10 mL pre-warmed to 37C

Muscle Fiber Dissection and Digestion

  • Place ~20 mL KRBH, ~20 mL DMEM and ~100 mL aCSF Media in a 37C water bath to warm.
  • Steriilze surgical tools for ~30 min and move mice to the procedure room.
  • Dissect FDB muscles from mice (see FDB Muscle Dissection from Mice). Place both muscles from a mouse in a single 12-well containing 1 mL of pre-warmed KRBH.
  • Weigh out enough collagenase to make 1 mL of a 4 mg/mL solution for each mouse. Add DMEM to make the 4 mg/mL solution
  • Gently transfer muscles to wells containing 1 mL of the collagenase solution.
  • Incubate at 37C for 1.5-2h to allow for digestion.
  • Gently transfer muscles to a new well containing 1 mL DMEM.
  • Pipet muscles up and down gently 5-10 times until the muscle is dissociated. If dissociation did not yield a sufficient number of fibers, digest for a further 15-30 minutes and repeat the process.
  • Try to pick out any obvious undigested material such as tendons using sterile tweezers.
  • Prepare XF24 plate by combining 100 uL matrigel with 100 uL media. Mix well by tapping.
  • Add 3 uL of this mixture to each well of a XF24 plate. Disperse the matrigel by hitting the plate against your hand to cover the surface.
  • Leave uncovered in the tissue culture hood for 30 minutes for the matrigel to dry.
  • Gently swirl the digested fibers and pipet 50 uL of the digested fiber mixture to each well.
  • Fibers should attach within 5 minutes. The assay can now be run, or cells can incubate overnight prior to the assay.

Mitochondrial Stress Test