Materials and Reagents

Syringe with needle 1 ml 25 G x 5/8 in. (0.5 x 16 mm) (BD, catalog number: 300014 )
Dishes 35 x 10 (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 153066 )
70 μm nylon strainer (Sinun Tech, catalog number: Polymer Screens )
FACS tubes (Corning, Falcon^TM, catalog number: 352054 )
Cells of interest (here murine bone marrow cells obtained from 8 weeks old mice)
Dulbecco’s phosphate-buffered saline (PBS^+/+) (Biological Industries, catalog number: 02-020-1A )
EDTA (5 mM final concentration in water, pH 7.4) (Avantor^® Performance Materials, J.T.Baker, catalog number: 8993-01 )
Wet ice
Antibodies to detect LT-HSCs by flow cytometry:

Sca-1 PEcy7 (clone D7) (Biolegend, catalog number: 108114 )
c-kit APC (clone 2B8) (Biolegend, catalog number: 105812 )
CD150 Brilliant Violet (clone TC15-12F12.2) (Biolegend, catalog number: 115922 )
CD48 Pasific Blue (clone HM48-1) (Biolegend, catalog number: 103418 )
EPCR PE (clone eBio1560) (Affymetrix, eBioscience, catalog number: 12-2012-82 )
Lineage: CD4 (clone GK 1.5), CD8a (clone 53-6.7), GR1 (clone RB6-8C5), B220 (clone RA3-682), Ter119 (clone TER-119), CD11b (clone M1/70)

Calcium chloride dihydrate (EMD Millipore, catalog number: 102382 )
Magnesium chloride hexahydrate (EMD Millipore, catalog number: 105833 )
HEPES buffer solution (Biological Industries, catalog number: 03-025-1B )
Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 14175095 )
Bovine serum albumin solution (10%, BSA) (Biological Industries, catalog number: 03-010-1B )
2.4-((N’-2-methylphenyl)ureido)-phenylacetyl-L-leucyl-L-aspartyl-L-valyl-L-prolyl-L-alanyl-L-alanyl-L-lysine (LDV-FITC) (R&D System, catalog number: 4577 )
Deionized water (DDW)
Gentian violet (Sigma-Aldrich, catalog number: 548-62-9 )
Acetic acid (Sigma-Aldrich, catalog number: 64-19-7 )
Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 30525-89-4 )
LDV medium (see Recipes)
LDV-FITC stock and working solutions (see Recipes)
Turk’s solution (see Recipes)
4% PFA (see Recipes)

Note: Flurochrome should be chosen according to the flow cytometry machine.

Equipment

Forceps (Kent Scientific, catalog number: INS700100-2 )
Scissors (Kent Scientific, catalog number: INS750046 )
Centrifuge (Eppendorf, model: 5810R )
Centrifuge swing-bucket rotor A-462 4 x 250 ml rectangular buckets (Eppendorf, catalog number: 5810709008 )
Adapters (Eppendorf, catalog number: 5810752000 )
Inverted light microscope (Olympus, model: CHK2-F-GS )
Note: This product has been discontinued by the manufacturer.
Hemocytometer (Sigma-Aldrich, Bright-Line^TM, catalog number: Z359629 )
Incubator 37 °C, 5% CO₂ (Thermo Fisher Scientific, Thermo Scientific^TM, model: 150i )
Cold room or refrigerator (4 °C)
Flow cytometer (i.e., Macs Quant instrument [Miltenyi, BergischGladbach, Germany] or BD LSR II flow cytometer)
2 L glass flask or bottle (Kimble Chase Life Science and Research Products, catalog number: 26500-2000 )
Plastic weigh boat (Sigma-Aldrich, catalog number: Z186856 )

Note: Color combinations can be adjusted to match the laser combinations available.

Software

FlowJo V10 software (Tree Star, optional)

Procedure

Obtain murine bone marrow cells
Note: All animal experiments have to be approved by local animal care and ethics authorities.
1. Sacrifice desired mice strain by CO₂ euthanization or cervical dislocation.
2. Extract the femurs, tibias and iliac crest bones using forceps and sharp scissors. Place the bones in a small dish supplemented with PBS^+/+on ice.
3. Flush total bone marrow cells from the bone cavity with 1-5 ml ice cold PBS (per mouse) using a 1 ml syringe and 25 G needle. Obtain single cell suspension by simply resuspending the cell solution using the same syringe.
  
  Figure 1. Process of flushing bone marrow out of murine bones. A. Femurs, tibias and iliac crest bones before flushing; B. Femurs, tibias and iliac crest bones after flushing; C. 1 ml syringe with its 25 G needle inserted in the tibia bone cavity.
4. Filter the cells by passing the cell solution through 70 μm nylon strainer to obtain a uniform single-cell suspension.
5. Centrifuge the cells (289.5 x g, 5 min) and resuspend in 1 ml LDV medium.
6. Count the cells by diluting the cells 1:10 with Turk’s solution. Count the cells under the inverted light microscope using the hemacytometer. From one mouse typically one will get 60-90 million cells from both femurs and tibias and iliac crest bones.
Determination of VLA-4 affinity using flow cytometry (LDV assay)
VLA-4 affinity can be examined on fresh bone marrow cells obtained from treated animals. Alternatively, cells can be obtained from untreated animals and treated in vitro with a desired reagent, in the presence of LDV medium, followed by the abovementioned detailed protocol.
Note: It is recommended to continue with the staining after isolation of the bone marrow cells.
1. Place 5 million bone marrow cells in a total volume 100 μl of LDV medium (see Recipes) in a FACS tube.
2. Add LDV-FITC to achieve a final concentration of 10 nM (see Recipes) and mix gently by pipetting, do not vortex.
3. To determine LDV-FITC non-specific binding, add EDTA to each LDV-FITC sample at a final concentration of 5 mM.
4. Incubate all the samples for 30 min at 37 °C.
5. After incubation, put the tubes immediately on wet ice and fix the cells by adding ice cold 4% PFA for 10 min (i.e., the reaction is in 100 µl of cells, thus add 4% PFA [1-2 ml] directly to the tube). Since that moment, cells need to be kept under cold conditions on wet ice. Avoid vortex along the procedure.
6. Wash the cells with 2 ml ice cold PBS^+/+.
7. Centrifuge the cells (289.5 x g, 5 min) and gently discard the supernatant.
8. Add 100 µl of ice cold PBS^+/+ and perform staining of the cell surface markers Lineage (1-2 µl per sample of each antigen), Sca-1 (2 µl/sample), c-Kit (2 µl/sample), CD150 (2 µl/sample), CD48 (1 µl/sample) and EPCR (2 µl/sample). These cell surface markers are only suggestive antigens to detect LT-HSCs, however, this can be adjusted according to the research question needs. Color combination should fit with FITC as LDV peptide is conjugated with the FITC fluorochrome.
9. Add the required antibodies into the cell samples and mix gently by pipetting (do not vortex), and incubate for 30 min on ice and protected from light.
10. Wash the cells with 1 ml ice cold PBS^+/+, centrifuge (289.5 x g, 5 min) and gently discard the supernatant.
11. Resuspend the cells in 300 μl PBS^+/+, do not vortex.
12. Read the samples immediately by flow cytometry and determine LDV binding to LT-HSC by analyzing the intensity of the FITC fluorochrome on gated Lineage^-/c-Kit⁺/Sca-1⁺/CD150⁺/CD48^-/EPCR⁺ cells.

Data analysis

Figure 2. VLA4 affinity measured by LDV probe binding to bone marrow EPCR⁺ SK (Sca1⁺ ckit⁺) SLAM (CD150⁺ CD48^-) cells. In black is the control cells treated with EDTA; in blue the EPCR+ cells gated on SLAM/SK; in pink the EPCR-cells gated on SLAM/SK.

For further analysis information concerning gating strategies and statistical analysis you can consult the article Gur-Cohen et al., 2015 at the following link: http://www.nature.com/nm/journal/v21/n11/full/nm.3960.html.

Recipes

LDV medium
1 mM CaCl₂
1 mM MgCl₂
20 mM HEPES, containing 1% BSA
Note: It is recommended to prepare stock solutions of 1 M CaCl₂ (dilute 1:1,000 directly in the medium) as well as 100 mM MgCl₂ (dilute 1:100 directly in the medium).
LDV-FITC stock and working solutions
Dissolve 1 mg LDV-FITC powder in 1 ml DDW (ddH₂O) according to the manufacturer's instructions (giving a stock concentration of 0.73 mM)
Note: It is recommended to divide the solution into small aliquots, avoiding repeated freeze/thaw cycles (the solution can be stored at -20 °C for up to 1 year).
To prepare the working LDV-FITC solution, dilute stock solution with DDW, reaching to a final concentration of 10 nM (for 10 nM working solution dilute 1:730 with DDW and take 1 µl into 100 µl cells supplemented with LDV medium)
Turk solution
50 mg gentian violet
5 ml acetic acid
495 ml DDW
Dissolve gentian violet in acetic acid and DDW
4% PFA
Weight out 40 g of powdered paraformaldehyde into a plastic weigh boat
Pour into a 2 L glass flask or bottle
Add 1 L DDW, a stir bar and allow to gently agitate while warming to 65 °C, for 5 min
Add one drop of 10 N KOH or 10 N NaOH base, the solution should then become clear
Allow the solution to cool to room temperature and adjust the pH to 7.3
Note: Smaller volumes can be stored at -20 °C for up to one year.

Acknowledgments

This study was supported by the Israel Science Foundation (851/13), the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine and FP7-HEALTH-2010 (CELL-PID 261387) (T.L.) and the DKFZ, Germany.

References

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