Mouse+Bone+Marrow+Isolation+Protocol

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
 * "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.
 * 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.
 * 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.
 * The robosep protocol is Mouse Progenitor Negative Selection 19756-MP & high recovery
 * The robosep protocol is Mouse Progenitor Negative Selection 19756-MP & high recovery

How many stem cells can be obtained?