Introduction
This protocol describes the isolation of cells from the marrow of bone [1,2]. As compared with whole blood, bone marrow yields a more complex set of cells, including hematopoietic stem cells–responsible for the production of leukocytes, erythrocytes, and thrombocytes–and stromal cells, such as endothelial cells, fibroblasts, macrophages, osteoblasts, osteoclasts, and adipocytes.

Materials


Procedure

Cell isolation

  1. Under sterile conditions, isolate bone marrow femurs from mice, and place into a sterile cell culture dish.
  1. Use forceps and small scissors to cut away the muscle and fibrous tissues from the bone. If muscles are still intact, wipe the bones with a Kimwipe saturated with 70% ethanol to remove any excess muscle fibers.
  1. Add 5–7 mL RPMI complete media to a new sterile cell culture dish. Prepare complete RPMI 1640 medium by supplementing RPMI 1640 medium with fetal bovine serum to a final concentration of 10%, 2 mM L-glutamine (if using medium not currently supplemented with GlutaMAX). Optional: Supplement media with 1% penicillin-streptomycin (5,000 units/mL).
  1. Cut both femur ends with a scalpel or sharp scissors so that both ends are clean.
  1. Attach a 25-gauge needle to a 10 mL syringe. Draw up 5 mL RPMI complete media into the syringe.
  1. Using forceps, hold the bone above the cell culture dish containing 5–7 mL RPMI complete media, and carefully flush out the marrow into the cell culture dish using the syringe containing 5 mL RPMI complete. Repeat flushing as necessary until the bone is white, which indicates that all the marrow has been removed. Repeat this step for all bones.
  1. Place a 70 µm cell strainer on top of a new 50 mL conical tube, held upright in a tube rack.
  1. Using a sterile serological pipet, transfer all marrow from cell culture dish into the strainer. Add more RPMI complete media to the cell culture dish and continue transferring any leftover marrow to the strainer.
  1. Using just the plunger from a 10 mL syringe (DO NOT TOUCH the black rubber end of the plunger; keep it sterile), mash the marrow contained in the 70 µm cell strainer in a downward circular motion. Verify there are no large pieces of bone marrow left.
  1. Rinse any leftover marrow from the plunger end with RPMI complete media into the strainer to collect as many cells as possible.
  1. Rinse strainer with 5–7 mL RPMI complete media. Remove the strainer and cap the conical tube containing the sample.
  1. Spin down cells at 600 x g for 4 minutes at 4°C. Discard the supernatant.
  1. Add about 5 mL RPMI complete media to the cell pellet, and pipet up and down to resuspend.
  1. Place a new 70 µm cell strainer on top of a new 50 mL conical tube in a rack.
  1. Strain the resuspended marrow sample through the cell strainer and into the conical tube. Count viable cells if desired.
  1. Resuspend cell sample to desired cell concentration with appropriate media.

Protocol tip:

After flushing the femur bones, they may be crushed by mortar and pestle for additional cells. Bone marrow can also be isolated from crushed pelvic bones and tibias.

Cell counting

  1. Lyse a small sample of the red blood cells with an RBC lysis buffer to easily count immune cells. Add 1 mL 1X RBC lysis buffer to 100 μL sample.
  1. Incubate at room temperature for 4–5 minutes with occasional shaking or rotating.
  1. Add 2–3 mL 1X PBS to stop the reaction.
  1. Spin down the cells at 600 x g for 4 minutes at 4°C. Discard the supernatant.
  1. Add about 500 μL complete RPMI to pellet, and pipet up and down to break the pellet.
  1. Count viable cells with a hemocytometer or the Invitrogen Countess 3 Automated Cell Counter.

Optional:

Proceed to immune cell isolation protocols using Invitrogen MagniSort Kits or Invitrogen Dynabead Kits or to cell stimulation protocols.


References

  1. Madaan A, Verma R, Singh AT et al. (2014) A stepwise procedure for isolation of murine bone marrow and generation of dendritic cells. J Biol Methods 1(1):e1.
  2. Liu X, Quan N (2015) Immune cell isolation from mouse femur bone marrow. Bio Protoc 5(20):e1631. PMID 27441207.