Authors
Xiaoyu Liu and Ning Quan
Summary
This protocol provides a simple and fast method for isolation of bone marrow immune cells (no erythrocytes) from mouse femurs with a yield of approximate 8 x 107 cells in 5 ml culture media (1.6 x 104 cells/µl). Further isolation or flow cytometric analysis might be required for study of specific immune cell types.
Introduction
The bone marrow is the site of hematopoesis and contains mixed population of blood cells including erythrocytes, granulocytes, monocytes, dendritic cells, lymphocytes and hematopoietic stem cells. The following protocol provides a simple and fast method for
isolation of bone marrow immune cells
Materials
1. Sterile paper towel
2. Sterile surgical pad (Direct Resource, catalog number: 19015742)
3. 23-gauge (or 25-/26-gauge) needle (BD Biosciences, catalog number: 305145)
4. 10 ml syringe (BD Biosciences, catalog number: 309604)
5. 70 µm nylon cell strainer (Falcon, catalog number: 352350)
Note: Currently, it is “Corning, Falcon®, catalog number: 11995-065”.
6. 50 ml conical tube (Falcon, catalog number: 21008-940)
Note: Currently, it is “Corning, Falcon®, catalog number: 21008-940”.
7. 5 ml syringe plunger (BD Biosciences, catalog number: 309646)
8. Adult mice (> 6 weeks, any strain) (e.g. C57BL/6)
9. Hank’s balanced salt solution (HBSS), no Calcium, no Magnesium, no phenol red (Life
Technologies, catalog number: 14175095)
Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 14175095”.
10. DMEM medium, high glucose, pyruvate, L-glutamine (Life Technologies, catalog
number: 11995-065)
Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 11995-065”.
11. 70% ethanol
12. Fetal bovine serum heat inactivated (FBS) (Sigma-Aldrich, catalog number: F9665)
13. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: 213330)
14. Potassium bicarbonate (KHCO3) (Sigma-Aldrich, catalog number: 237205)
15. Disodium edetate (Sigma-Aldrich, catalog number: D2900000)
16. RBC lysis buffer: 0.16 M NH4Cl, 10 mM KHCO3, and 0.13 mM EDTA, dissolved in sterile H2O and stored at 4 °C. For 500 ml, 4.28 g NH4Cl, 0.5 g KHCO3, 0.024 g Disodium EDTA It is recommended to prepare fresh RBC lysis buffer for the experiment. RBC lysis buffer will be stable at 4 °C for at least 1 month.
17. DMEM medium: DMEM medium, high glucose, pyruvate, L-glutamine supplemented with 10% FBS. Stored at 4 °C
Equipment
1. Blunt-end sterile scissors (Thermo Fisher Scientific, Fisher Scientific, catalog number:
08-950)
2. Sharp sterile scissors (Thermo Fisher Scientific, Fisher Scientific, catalog number:
08-940)
3. Sterile forceps (Thermo Fisher Scientific, Fisher Scientific, catalog number: 08-890)
4. Hausser™ Levy™ Hemacytometer Chamber Set (Thermo Fisher Scientific, Fisher
Scientific, catalog number: 02-671-55A) or coulter Z2 cell and particle counter
(Beckman Coulter, catalog number: 383550)
5. Refrigerated centrifuge
6. Sterile culture hood
7. CO2 rodent euthanasia chamber
Procedure
1. Euthanize the mouse with CO2 and place mouse onto a sterile surgical pad in a sterile
hood. Sterilize the mouse abdomen area and skin of hindlimbs with 70% ethanol.
2. Open the abdominal cavity with blunt-end sterile scissors and remove the surface
muscles and find the pelvic-hip joint.
3. Cut off the hind leg above the pelvic-hip joint with sharp sterile scissors. Cut
off the tibia from the hind leg below the knee joint with sharp sterile scissors.
4. (Optional) If higher yield of bone marrow cells is needed, tibia can also be used for
bone marrow cell isolation. Cut at the tibia ankle joint to dissect the tibia. The following
procedures can be applied to both femur and tibia.
5. Remove the muscles and residue tissues surrounding the femur with sterile forceps
and scissors.
6. Cut the femurs at both ends with sharp sterile scissors. Use a 23-gauge
(some literature suggests 25-or 26-gauge) needle and a 10 cc syringe filled with
ice-cold HBSS to flush the bone marrow out onto a 70 µm nylon cell strainer placed in
a 50 ml Falcon conical tube. Use all the 10 ml HBSS or until the flow
through turns white.
7. (Optional) In case some residue bone marrow cells could not be flushed off (very few
bone marrow visible in the flow through or the yield is significantly less, e.g. < 1 x 107
cells), scrape the inner surface of the femur with the needle and flush with extra ~5 ml
HBSS.
8. Smash the bone marrow through the cell strainer with a 5 ml plunger. Wash
the strainer with another ~5 ml HBSS.
9. Centrifuge cells at 1,500 rpm for 7 min at 4 °C. Discard the supernatant and blot on
paper towel.
10. Resuspend the cell pellet with 1 ml RBC lysis buffer (for each mouse). Incubate for 5
min at room temperature or 2 min at 37 °C, and neutralize the lysis buffer by adding 5
ml FBS.
11. Centrifuge cells at 1,500 rpm for 7 min at 4 °C. Discard the supernatant and blot on
paper towel. Resuspend the cell pellet with appropriate media for the next step of
assay such as 5 ml DMEM medium containing 10% FBS. Cells are then placed on ice.
12. Count the bone marrow cells with a hemocytometer or a Beckman Z2 coulter counter.
Cells are ready for assays or culture. Cells can stay viable on ice for at least 5 h. It is
recommended to perform the experiment (culture or assays) right after isolation for
best results.
Anticipated results
The yield of bone marrow immune cells (no erythrocytes) from mouse femurs is approximate 8 x 107 cells in 5 ml culture media (1.6 x 104 cells/µl).
References
1. Madaan, A., Verma, R., Singh, A. T., Jain, S. K. and Jaggi, M. (2014). A stepwise
procedure for isolation of murine bone marrow and generation of dendritic cells. J Biol
Methods 1(1): e1.
2. Weischenfeldt, J. and Porse, B. (2008). Bone marrow-derived macrophages (BMM):
Isolation and applications. CSH Protoc 2008: pdb prot5080.
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