Isolation of Single Fibers for Seahorse Assays

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

Place ~20 mL KRBH, ~20 mL DMEM and ~100 mL aCSF Media in a 37C water bath to warm.
Sterilize 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.