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.
- Cell culture dishes (e.g., Thermo Scientific Nunc EasYDish Dishes, 100 mm, Cat. No. 150466)
- Lab wipes (e.g., Fisher Scientific Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers, 1-PlyFisher, Fisher Scientific Cat. No. 06-666)
- Ethanol (e.g., Absolute Ethanol, 200 Proof, Molecular Biology Grade, Cat. No. T038181000CS)
- RPMI Complete Media
- RPMI 1640 medium (e.g., Gibco BenchStable RPMI 1640, Cat. No. A4192301)
- 2 mM L-glutamine (e.g., Gibco L-Glutamine, 200 mM, Cat. No. 25030149)
- Fetal bovine serum (e.g., Gibco Fetal Bovine Serum, Cat. No. 26140087)
- Optional: Penicillin-streptomycin (e.g., Gibco Penicillin-Streptomycin (5,000 U/mL), Cat. No. 15070063, or Gibco Penicillin-Streptomycin (10,000 U/mL), Cat. No. 15140122)
- Scalpel and blades
- 5–10 mL syringe (e.g., Fisherbrand Sterile Syringes for Single Use, 10 mL, Cat. No. 14-955-459)
- 25-gauge needle (e.g., Fisher Scientific BD General Use and PrecisionGlide Hypodermic Needles, Cat. No. 14-826-49)
- 70 μm cell strainers (e.g., Fisherbrand Sterile Cell Strainers, Cat. No. 22-363-548)
- 50 mL conical tubes (e.g., Thermo Scientific Nunc 50 mL Conical Sterile Polypropylene Centrifuge Tubes, Cat. No. 339652)
- Red blood cell (RBC) lysis buffer (e.g., Invitrogen eBioscience 1X RBC Lysis Buffer, Cat No. 00-4333-57 or Invitrogen eBioscience 10X RBC Lysis Buffer (Multi-species), Cat. No. 00-4300-54)
- Cell counter (e.g., Invitrogen Countess 3 Automated Cell Counter, Cat. No. AMQAX2000)
- Phosphate-buffered saline (PBS) (e.g., Gibco PBS (10X), pH 7.4, Cat. No. 70011069)
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.
- 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.
- Liu X, Quan N (2015) Immune cell isolation from mouse femur bone marrow. Bio Protoc 5(20):e1631. PMID 27441207.
For Research Use Only. Not for use in diagnostic procedures.