Materials and Reagents

Gauze sponges (Fisher Scientific, Fisherbrand^TM, catalog number: 22-415-468 )
21 G needles
3 ml syringes
70 μm strainer (Fisher Scientific, Fisherbrand^TM, catalog number: 22-363-548 )
1.5 ml microcentrifuge tube (NEST Biotechnology, catalog number: 615601 ), 15 ml tubes (BioExpress, GeneMate^TM, catalog number: C-3394-1 ), and 50 ml conical tubes (BioExpress, GeneMate^TM, catalog number: C-3394-4 )
Other antibodies:
APC-anti-CD31 (Thermo Fisher Scientific, eBioScience^TM, catalog number: 17-0311-82 )
PE-anti-CD51 (BD, BD Pharmingen^TM, catalog number: 551187 )
PE-Cy7-anti-CD45 (BD, BD Pharmingen^TM, catalog number: 552848 )
Biotin-Sca1 (BD, BD Pharmingen^TM, catalog number: 553334 )
Streptavidin APC-Cy7 (BD, BD Pharmingen^TM, catalog number: 554063 )
CXCL12 detection antibodies: H/M CXCL12/SDF-1 Fluorescein (FITC) MAb (Clone 79018) (R&D Systems, catalog number: IC350F )
70% (v/v) ethanol
Hank’s Balanced Salt Solution (HBSS; 1x) (GE Healthcare, Hyclone^TM, catalog number: SH30588.02 )
BSA (Sigma-Aldrich, catalog number: A7906 )
Type I collagenase (Worthington, Lakewood, NJ)
DMEM (ATCC^®, catalog number: 30-2002^TM )
Fixation/Permeabilization Solution Kit with BD GolgiStopTM (BD, BD Cytofix/Cytoperm™ Plus, catalog number: 554715 )
HBSS staining buffer (see Recipes)

Equipment

Pipettes
Scissors
Tweezers
Curved forceps
Tabletop centrifuge (SORVALL Legend RT)
Hemocytometer
FACSAria I

Procedure

Preparing single cell suspension from long bones and osteoblast sorting
1. Euthanize the mouse by CO₂ asphyxiation and confirmed death by cervical dislocation.
2. Sterilize the body with 70% (v/v) ethanol.
3. Make incisions around the connection between hind limbs and trunk using scissors and tweezers. The whole skin is then removed from the hind limbs by pulling toward the cutting site of the claw (Figure 1A). Carefully remove all the muscles from the long bones (femur and tibia) using dissecting scissors and curved forceps. Excise the long bone and transfer onto sterile gauze.
4. Clean soft tissue as thoroughly as possible around the bone (Figure 1B).
  
  Figure 1. Preparation of mouse long bones. A. Exposure of hind legs. B. Cleaned long bones (tibia and femur) after removing attached muscles.
5. Cut off the epiphyses.
6. Fill a 21-gauge needle and 3 ml syringe with 1 ml cold HBSS supplemented with 0.5% BSA and use a syringe to flush out bone marrow (BM) three times (Figure 2A).
7. Cut the bone into small pieces of approximately 1-2 mm² using scissors (Figure 2B).
  
  Figure 2. Preparation of long bone fragments. A. Cleaned long bones after flushing out bone marrow; B. Prepared bone fragments.
8. Digest bone fragments obtained from 2 sets of tibias and femurs per mouse in 2 ml HBSS buffer supplemented with collagenase I (3 mg/ml in DMEM) in a 50 ml conical tube for 1 h by shaking (110 rpm) at 37 °C.
9. Add 3 volume of DMEM (6 ml) supplemented with 10% FBS to terminate collagenase I activity.
10. Strain the cell solution through a 70 μm strainer in a 50 ml conical tube.
11. Spin down at 250 x g for 5 min at 4 °C.
12. Remove supernatant
13. Wash the cells with 2 ml of HBSS buffer and spin down at 250-400 x g for 5 min at 4 °C.
14. Discard supernatant and resuspend the cells in 1 ml HBSS buffer.
15. Determine cell number. Count cells using a hemocytometer.
16. Spin down at 250-400 x g for 5 min at 4 °C.
17. The cells are ready for preparing for osteoblast sorting and CXCL12 staining.
18. Prepare antibody cocktails in HBSS buffer using the following antibodies: FITC- lineage markers (T cells: CD4, CD8; B cells: B220; erythrocytes: Ter119; granulocytes and monocytes: CD11b, and Gr-1), APC-CD31 (endothelial cell), PE-CD51, PE-Cy7-Sca1. Use 100 µl of HBSS buffer containing 1 µl of each antibody for 1 x 10⁶ cells.
19. Resuspend cells from Step A17 in antibody mixture on ice for 20 min.
20. Wash cells twice each with 200-500 µl cold HBSS buffer, spinning down at 250-400 x g for 5 min at 4 °C in between washes.
21. Sort cells with FACSAria I. Osteoblasts are sorted by gating on lin^- (CD4, CD8, B220, Ter119, CD11b, Gr-1) CD31^-CD51⁺Sca1^- population (Figures 3A and 3B).
  
  Figure 3. Sorting of osteoblasts. A. Gating strategy of lin^- (CD4, CD8, B220, Ter119, CD11b, Gr-1) CD31^- population; B. Gated osteoblasts (lin^- CD31^-CD51⁺Sca1^-).
CXCL12/SDF-1 Intracellular Staining

Resuspend cells from Step A17 from procedure A in HBSS buffer in a 15 ml Falcon tube. Add biotin-Sca1 antibody and leave cells on ice for 20 min. Use 100 µl of HBSS buffer containing 0.5 µl of antibody for 1 x 10⁶ cells.
Wash cells twice each with 200-500 µl HBSS buffer.
Spin down at 250 x g for 5 min at 4 °C. Resuspend cells in HBSS buffer and add APC-CD31, PE-CD51, PE-Cy7-CD45, and Streptavidin APC-Cy7 antibodies, and then incubate on ice for 20 min.
Wash twice each with 1 ml HBSS buffer. Spin down cells at 250 x g for 5 min.
Fix and permeabilize cells by resuspending cells in 250 µl of Fixation/Permeabilization solution (included in the BD Cytofix/Cytoperm^TM Plus) for 15-20 min at 4 °C.
Note: Cell aggregation can be avoided by gently vortexing prior to the addition of the Fixation/Permeabilization solution.
Wash cells twice each with 1 ml of 1x Perm/Wash^TM solution (included in the BD Cytofix/Cytoperm^TM Plus), pellet cells by centrifugation at 250 x g for 5 min at 4 °C, and remove supernatant.
Note: Perm/Wash^TM solution is required in washing steps to maintain cells in a permeabilized state.
Stain intracellular CXCL12 by adding 10 μl of FITC-CXCL12 in 90 μl 1x Perm/Wash^TM solution and vortex gently.
Incubate cells for 30 min at room temperature in a dark room.
Wash cells twice each with 1 ml 1x Perm/Wash^TM Buffer. Spin down cells at 250 x g for 5 min at 4 °C.
Resuspend cells in 200 μl HBSS buffer for flow cytometric analysis (see Figure 4 below).
Note: For a negative control, a separate set of cells should be stained with an isotype control antibody.

Data analysis

From wild type (WT) mice long bones, osteoblasts are readily isolated from the bone marrow stroma after brief digestion with collagenase I (Figure 4A). In comparison, osteoblasts are decreased in the marrow stroma from mice developing T-ALL driven by activated Notch1 (Figure 4B). CXCL12 expression by intracellular staining and flow analysis shows 52% decrease in leukemia mice osteoblasts compared to normal controls (Figure 4C).

Figure 4. Sorting and CXCL12 staining of bone marrow osteoblasts. Bone marrow stroma cells were prepared from WT or leukemia-developing mice. Staining of marrow osteoblast (OB) (lin^-CD31^- CD51⁺Sca-1^-) (A and B) by gating on the lineage^-CD31^-popualtion. Intracellular expression of CXCL12 was displayed by mean fluorescence intensity (MFI) using isotype control or anti-CXCL12 antibody (C).

Recipes

HBSS staining buffer
Hank’s Balanced Salt Solution (HBSS; 1x) supplemented with 0.5% BSA

Acknowledgments

This work was supported by grants from American Cancer Society LIB-125064 (L. Zhou) and NIH HL103827 (L. Zhou). No potential conflicts of interest were disclosed.

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