Difference between revisions of "Isolation of Single Fibers for Seahorse Assays"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (wrote initial protocol) |
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==Muscle Fiber Dissection and Digestion== | ==Muscle Fiber Dissection and Digestion== | ||
* Place ~20 mL KRBH, ~20 mL DMEM and ~100 mL aCSF Media in a 37C water bath to warm. | * 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. | * 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 | * 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 | ||
Latest revision as of 17:06, 13 June 2017
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.
- 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.
