Immune Cell Isolation from Mouse Femur Bone Marrow

Abstract

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 (no erythrocytes) from mouse femurs with a yield of approximate 8 × 10⁷ cells in 5 ml culture media (1.6 × 10⁴ cells/μl). Further isolation or flow cytometric analysis might be required for study of specific immune cell types.

Materials and Reagents

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 (see Recipes)

17. DMEM medium (see Recipes)

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 (Figure 1).

2. Open the abdominal cavity with blunt-end sterile scissors and remove the surface muscles and find the pelvic-hip joint (Figure 2).

3. Cut off the hind leg above the pelvic-hip joint with sharp sterile scissors (Figure 3). Cut off the tibia from the hind leg below the knee joint with sharp sterile scissors (Figure 4).

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 (Figure 5).

6. Cut the femurs at both ends with sharp sterile scissors (Figure 6). 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 (Figure 7). 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 × 10⁷ cells), scrape the inner surface of the femur with the needle and flush with extra ~5 ml HBSS (Figure 8).

8. Smash the bone marrow through the cell strainer with a 5 ml plunger (Figure 9). 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 (Figure 10).

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.

Figure 1.

Sterilization of mouse abdomen area and skin of hindlimbs

Figure 2. Find the pelvic-hip joint.

Bone anatomy reference: http://www.informatics.jax.org/cookbook/figures/figure41.shtml

Figure 3.

Cut off the hind leg above the pelvic-hip joint

Figure 4.

Cut off the tibia at knee joint

Figure 5.

Remove the muscles and residue tissues surrounding the femur

Figure 6.

Cut femurs at both ends

Figure 7.

Flush the bone marrow onto the cell strainer with HBSS

Figure 8.

(Optional) Scrape the inner surface of femur with needle

Figure 9.

Smash the bone marrow cells through the cell strainer with a 5 ml plunger

Figure 10.

Cell pellet before RBC buffer resuspension

Recipes

1. 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.

2. DMEM medium

   • DMEM medium, high glucose, pyruvate, L-glutamine supplemented with 10% FBS

   • Stored at 4 °C