View source for Isolation of Single Fibers for Seahorse Assays


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# [[https://www.thermofisher.com/order/catalog/product/21063029 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==