Materials and Reagents

Tissue culture 6-well plates (Corning, Costar^®, catalog number: 3516 )
Nunc^TM 8.8 cm² Petri dish (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 153066 )
1 ml slip tip Sub-Q syringe with disposable 26 G x 5/8 inch needle (BD, catalog number: 309597 )
Autoclaved pipet tips:
1. 200-1,000 µl blue, gilson tip (Greiner Bio One, catalog number: 740290 )
2. 200 µl universal tip (Neptune Scientific, catalog number: 2100.N )
3. 0.5-10 µl pipet tips (Corning, Axygen^®, catalog number: T-300 )
FACS tubes (Corning, Falcon^®, model: 352054 )
Sterilized 40 µm pore-sized nylon mesh (Sinun Tech, catalog number: Plymer Screends ) (see Recipes)
Mice
Fibronectin (Sigma-Aldrich, catalog number: F0895 )
Human CXCL12 (human SDF-1 alpha) (reprokine^TM, catalog number: RKP48061 ) or (PeproTech, Rocky Hill, NJ, USA)
BSA (Sigma-Aldrich, catalog number: A9647 )
Lymphoprep^TM Ficoll (STEMCELL Technologies, catalog number: 07861 )
Antibodies:
1. EPCR PE (Affymetrix, eBioscience, catalog number: 12-2012-82 ) or EPCR PerCP-eFluor 710(Affymetrix, eBioscience, catalog number: 46-2012-80 ) or EPCR Biotin (Affymetrix, eBioscience, catalog number: 13-2012-82 ) combined with streptavidin-PE (BioLegend, catalog number: 405203 )
2. Sca-1 PE (BioLegend, catalog number: 108108 ) or Sca-1 PE/Cy7 (BioLegend, catalog number: 108114
3. c-Kit APC (BioLegend, catalog number: 105812 )
4. Lineage antibodies
  Note: This is a set of antibodies that target the antigens i-vi listed below which are cell lineage markers. In mice these markers do not occur on stem and progenitor cells.
  1. CD8a FITC (BioLegend, catalog number: 100706 )
  2. CD4 FITC (BioLegend, catalog number: 100406 )
  3. GR1 FITC (BioLegend, catalog number: 108406 )
  4. B220 FITC (BioLegend, catalog number: 103206 )
  5. TER-119 FITC (BioLegend, catalog number: 116206 )
  6. CD11b FITC(BioLegend, catalog number: 101206 )
  7. Or Lineage Cocktail-Biotin (Miltenyi Biotec, catalog number: 130-092-613 ) combined with streptavidin-FITC (BioLegend; catalog number: 405202 )
    Note: The combination of fluorophores used for the experiment needs to be adjusted according to the experimental demands.
1x Dulbecco’s phosphate buffered saline without calcium and magnesium (PBS^-/-) (generated from 10x PBS, see Recipes) (Biological Industries, catalog number: 02-023-5A )
Roswell park memorial institute (RPMI) 1640 medium ([+] 300 mg/L L-glutamine, [+] 25 mM HEPES) (Thermo Fisher Scientific, Gibco^TM, catalog number: 52400025 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 12657-029 )
Penicillin-streptomycin (Pen-Strep) solution (Biological Industries, catalog number: 03-031-1B )
L-glutamine solution (Biological Industries, catalog number: 03-020-1B )
Gentian violet (Sigma-Aldrich, catalog number: G2039 )
Acetic acid (Sigma-Aldrich, catalog number: ARK2183 )
0.1 M sodium azide solution (Sigma-Aldrich, catalog number: 0 8591 )
Acetone (Sigma-Aldrich, catalog number: 40289 )
Note: This product has been discontinued.
Ethanol (Sigma-Aldrich, catalog number: 24103 )
Note: This product has been discontinued.
Cell dissociation solution (Sigma-Aldrich, catalog number: C5914 )
ddH₂O
Coating solution (see Recipes)
Blocking solution with 2% (m/v) BSA (see Recipes)
Complete RPMI medium (see Recipes)
Turk’s solution (see Recipes)
FACS buffer (see Recipes)

Equipment

Heracell^TM 150i CO₂ incubator (Thermo Fisher Scientific, Thermo Scientific^TM, model: 150i CO₂ incubator)
Autoclave
Scissors
Forceps
Refrigerator (4° C)
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 )
Finnpipette model 4500 single channel pipette:

0.5-10 μl (Thermo Fisher Scientific, Thermo Scientific, catalog number: FA-10R )
5-40 μl (Thermo Fisher Scientific, Thermo Scientific, catalog number: FA-40R )
20-200 μl (Thermo Fisher Scientific, Thermo Scientific, catalog number: FA-200R )
100-1,000 μl (Thermo Fisher Scientific, Thermo Scientific, catalog number: FA-1000R )

Inverted light microscope (Olympus, model: CHK2-F-GS )
Note: This product has been discontinued by the manufacturer.
Counting chamber/hemocytometer (Reichert Bright-Line) (Sigma-AIdrich, Bright-Line^TM, model: Z359629 )
Flow cytometer (model optional):
1. MACSQuant VYB instrument (Miltenyi Biotec, model: 130096116 )
2. FACSCalibur instrument (BD)
3. FACS LSRII instrument (with all four fixed-aligned lasers) (BD)

Software

CellQuest software
FACSDiva software
FlowJo (Tree Star or V10) or MacsQuant

Procedure

Coating and preparation
1. Coat tissue culture 6-well plates with 25 µg/ml fibronectin and 2.5 µg/ml CXCL12 overnight at 4 °C. For the coating, it is recommended to prepare a mix of fibronectin and CXCL12 in PBS^-/- (coating medium) and to calculate the amount needed so that each well will be covered with 500 µl coating medium. Fibronectin stock (1 mg/ml) and CXCL12 (50 µg/ml) were stored at -20 °C.
2. After overnight incubation, discard the coating medium gently and wash the plates with 1 ml PBS^-/- for a few seconds. Repeat washing 3 times.
3. Perform blocking with 2% BSA in PBS^-/- for 30 min at room temperature. Blocking solution should cover each well of the plates, and therefore it is recommended to use a minimum of 500 µl blocking solution for each well.
4. Discard the blocking solution gently and wash plates with 1 ml PBS^-/- for a few seconds while tilting the plates slowly. Repeat washing 3 times.
5. Plates are ready to be seeded with BM mononuclear cells.
Obtaining bone marrow mononuclear cells
1. Sacrifice desired mice by CO₂ euthanasia or cervical dislocation.
2. Extract the femurs, tibias and pelvises using forceps and sharp scissors. Place the bones on a small dish supplemented with PBS^-/-.
3. Flush total bone marrow cells from the bone cavity with 1-2 ml PBS^-/- (per mouse) using a 1 ml syringe and 16 G needle (Figure 1). Obtain single cell suspension by resuspending the cell solution using the same syringe.
  
  Figure 1. Process of flushing bone marrow out of murine tibia. The red color of the extracted bones indicates the presence of the BM (A). Drilling the needle into the bone and flushing the BM with PBS out of the bone (B) until the bones look white enough (C).
4. Carefully pipet (very slowly) ficoll to the bottom of each tube with a dilution ratio of 1:2 to the bone marrow cells (i.e., if you have a total of 2 ml bone marrow cells in PBS, pipette 1 ml ficoll to the bottom of the tube) (Figures 2A and 2B).
  
  Figure 2. Process of MNC isolation from BM. Ficoll is added to the bottom of the tube underneath the suspended BM (A) forming a lower clear ficoll phase and an upper cell suspension phase (B). After centrifugation the two phases are separated by thin white phase consisting of MNCs (C) which is more prominent the more BM is used for the isolation (D).
5. Centrifuge the cells at 652 x g for 25 min, without a brake (important) at room temperature (mononuclear cells will not be separated if the centrifuge is cold).
6. Carefully remove the tubes from the centrifuge. At the middle phase a fine white ring-like shape should form, containing the bone marrow mononuclear cells (Figures 2C and 2D)
7. Carefully, discard the fluids on top of the ring with a pipet, while avoiding touching the mononuclear fraction. Collect the ring of cells with 200 µl pipette tip into a new FACS tube.
8. Wash the collected mononuclear cells with 2 ml PBS^-/- and centrifuge the cells at 452.8 x g for 5 min.
9. Discard the supernatant and resuspend the cell pellet in 1 ml complete RPMI (see Recipes).
10. Count the cells by diluting the cells 1:10 with Turk’s solution. Count the cells under the inverted light microscope using a hemocytometer. From one mouse typically one will get 20-40 million mononuclear cells.
Adhesion assay (Figure 3)
1. Seed bone marrow mononuclear cells at a density of 5 x 10⁶ cells per 1 ml complete RPMI medium.
2. Allow the cells to adhere to the coated plates for 2 h at 37 °C in an incubator.
3. Collect non-adherent cells to a new FACS tube by gently rinsing each well with complete RPMI medium. Make sure not to touch the bottom and only add RPMI by placing the pipet tip against the wall of each well.
4. Gently wash the plates 3 times with PBS^-/- by tilting the plates back and forth. Make sure not to touch the bottom and only add PBS^-/-by placing the pipet tip against the wall of each well.
5. To collect the adherent cells, incubate the plates with cell dissociation buffer for 10 min at 37 °C in the incubator.
6. Monitor the cells under the inverted microscope. If cells are still attached to the bottom, resuspend the cells by using pipet and incubate the plate for additional 5-10 min in the presence of the cell dissociation buffer.
7. Once the cells are detached from the well, vigorously rinse each well using a pipet and collect the adherent fraction to a new FACS tube. Subsequently, add 1 ml ice cold PBS^-/- to each well, rinse thoroughly and collect the fluids into the adherent cell fraction to a different FACS tube.
8. Monitor the cells under the inverted microscope. If additional cells remained on the plate, vigorously rinse each well again with ice cold PBS^-/- and keep collecting the fluids into the adherent cell fraction.
9. Centrifuge the adherent and nonadherent cell fractions at 452.8 x g, for 5 min.
10. Discard the supernatant carefully.
11. Add 100 µl PBS to each pellet. The cells are now ready to be stained and analyzed.
FACS staining and analysis (Figure 4)
1. Add 1 µl of each lineage antibody (CD8a FITC, CD4 FITC, GR1 FITC, B220 FITC, TER-119 FITC, CD11b FITC) and 2 µl Sca1 PeCy7, cKit APC and EPCR PE antibodies to remaining solution on the bottom of each tube.
  Note: Do not forget to include a negative control in order to be able to distinguish between the autofluorescence and positive signal during the flow cytometry.
2. Resuspend the pellets by vortexing the tubes briefly.
3. Leave the samples at 4 °C for 30 min.
  Note: If biotin-coupled antibodies are used, resuspend the cells in 1 ml PBS after the incubation at 4 °C and repeat the steps from C9 until this step. At step D1 add 1 µl of FITC-coupled streptavidin to the remaining solution of each tube.
4. Resuspend each sample in 1 ml FACS buffer and filter through a 40 µm mesh into a new tube to avoid clots during flow cytometry.
5. Centrifuge the sample at 452.8 x g for 5 min.
6. Discard the supernatant carefully, add 300 µl of FACS buffer to each sample and resuspend the cells by vortexing.
7. Read the samples using a flow cytometer. Cell populations are analyzed with a FACSCalibur instrument with CellQuest software, with a MacsQuant instrument or with a FACS LSRII instrument with FACSDiva software. Please consider that the combination of some fluorophores may cause an overlap in signals within the flow cytometer. Therefore, to set good compensation is mandatory.
8. Data are analyzed with FlowJo (Tree Star or V10) or MacsQuant according to the gating strategy illustrated in Figure 1 (see Data analysis section as well):
  1. Gate for live cells using forward (FSC) and sideward scatter (SSC).
  2. Gate for lineage negative cells using SSC and the channel for the fluorophore coupled to the lineage antibodies (Figure 5).
  3. Gate for SK cells selecting the c-Kit positive and Sca-1 positive cell population (Figure 5).
  4. Create a histogram overlay displaying the EPCR signals of the LSK population within the negative control, the adherent and nonadherent fractions.
    
    Figure 3. Diagramm showing steps of adhesion assay procedure (Procedure C)
    
    Figure 4. Diagramm showing steps of FACS staining procedure (Procedure D)
    
    Figure 5. Gating strategy of EPCR positive bone marrow LSK cells (adapted from Supp. Figure 5a of Gur-Cohen et al., 2015)

Data analysis

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

1x PBS^-/-
Add 50 ml of 10x PBS to 450 ml of dH₂O
Coating solution
1x PBS with 25 µg/ml fibronectin and 2.5 µg/ml CXCL12
Blocking solution with 2% (m/v) BSA
Add 2 g of BSA per 100 ml 1x PBS
Complete RPMI medium
88% RPMI 1640 medium
10% FBS
1% Pen-Strep solution
1% L-glutamine solution
Turk’s solution
50 mg of gentian violet
5 ml of acetic acid
495 ml of ddH₂O
Dilute gentian violet in acetic acid and ddH₂O
FACS buffer
Note: Mix this buffer in a plastic bottle.
450 ml ddH₂O
18 µl sodium azide
50 ml 10x PBS
5 ml FBS
Sterilized 24 µm pore-sized nylon mesh

Cut nylon mesh into approx. 3 x 3 pieces
Incubate the meshes for 20 min at room temperature in acetone under the chemical hood
Incubate the meshes for 20 min at room temperature in absolute ethanol
Let the meshes dry overnight
Sterilize the meshes by autoclaving them at 121 °C for 60 min

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.

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