Mouse Bone Marrow Isolation Protocol

2015-01-13

azim58 - Mouse Bone Marrow Isolation Protocol

Sources

* much of this protocol derives from Current protocols in Immunology:
  The Isolation and Characterization of Murine Macrophages, Basic
  Protocol 2
  (http://www.currentprotocols.com/WileyCDA/CPUnit/refId-im1401.html) as
  well as from
* A protocol for isolation and culture of mesenchymal stem cells from
  mouse bone marrow
  (http://www.nature.com/nprot/journal/v4/n1/full/nprot.2008.221.html)
* There are also tips on bone marrow procedures in the EasySep Mouse
  Hematopoietic Progenitor Cell Enrichment Kit
* Shen has a protocol from Mayo as well
  o "F:\kurt\storage\CIM Research
    Folder\DR\2013\5-26-13\wiki_download\bone marrow preparation protocol
    from Mayo.pdf"

Mouse Bone Marrow Isolation Protocol

Materials

* scissors
* forceps
* avertin
* syringe (3 mL is probably appropriate)
* needles (25-G)
* media (complete DMEM)
* Clorox Disinfecting Wipe?
*

* Anesthesize mouse. We typically use avertin (300 uL of 0.05 mg/mL
  diluted in H20?) injected intraperitoneally (i.p.) with 27.5 guage
  needle
* Euthanize mice by rapid cervical dislocation (UNIT 1.8). Using
  aseptic technique (surgical equipment should be cleaned with ethanol
  and the surgery should be performed in a hood), peel the skin from the
  top of each hind leg and down over the foot. Cut off the foot alongwith
  the skin and discard. Cut off the hind legs at the hip joint with
  scissors, leaving the femur intact. When cutting, try to cut at the
  joints to avoid cutting the bone open.
* Remove excess muscle from legs by holding end of bone with forceps
  and using scissors or hands to push muscle downward away from forceps.
  o Alternatively, excess muscle can be removed from the legs by
    holding the end of the bone with a piece of Clorox Disinfecting Wipe
    and pushing muscle away from bones with fingers.

* Place thigh bone into media such as complete DMEM media. The rest of
  the procedure could be performed in a tissue culture room instead of
  the mouse facility.
* Cut off the top and bottom of the bone.
* Attach 3-ml syringe to 25-G needle and fill with cold sterile wash
  medium (Complete DMEM or Dulbecco’s phosphate-buffered saline without
  calcium and magnesium). Insert syringe into the spongy bone exposed by
  removal of the growth plate. Flush the marrow plug out of the cut end
  of the bone with 1 mL of complete media and collect in a 10 mL tube or
  a petri dish with media. May want to keep things cold during this
  procedure.
* Pipette the bone marrow clumps up and down until the solution becomes
  mostly homogenous.
* Filter the suspension through a filter mesh (possibly 40 um or 70 um)
  to remove any bone spicules or muscle and cell clumps.
* May want to obtain a cell count.
* May want to centrifuge cells 10 min at 500 × g, room temperature.,
  but this may not be necessary.
  o Discard supernatant. Resuspend cell pellet in complete medium or
    Robosep buffer by tapping tube and pipetting up and down. You may
    want a final concentration appropriate for the ROBOSEP (volume of 500
    uL - 8.0 mL at a concentration of 1*10^8 cells/mL up to 8.0*10^8
    cells)

* May want to use Robosep with EasySep Mouse Hematopoietic Progenitor
  Cell Enrichment Kit to purify stem cells.
  o The robosep protocol is Mouse Progenitor Negative Selection
    19756-MP & high recovery

How many stem cells can be obtained?

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