Purification of Lin-, c-kit+, Sca-1+ bone marrow cells for Culture

MATERIALS

• Media:

• Heat inactivated FBS (56°C x 30 min)

• PBS + 2% heat-inactivated FBS

• IMDM + 20% heat-inactivated FBS

• Viral transduction: Iscove's + 20% heat inactivated FBS,

• plus 100 ng/mL each of SCF, IL-6, and Flt-3L, plus 10 ng/mL IL-11.

• CFU assays: aMEM +30% FBS, 1% deionized BSA, 1.2% methylcellulose, 0.1mM ßME.

• plus 3 U/mL Epo, 50 ng/mL Tpo, 100 ng/mL SCF, 100 ng/mL IL-6, ? ng/mL IL-3.

• 
  

• Antibodies for Lineage Depletion: (Pharmingen)

  T-cell: CD2 IgG2b, 01171D	B-cell: B220 (CD45R) IgG2a, 01121A
  CD3 IgG2b, 28001D	Monocytic: Mac1 (CD11b) IgG2b, 01711A
  CD5 IgG2a, 01031D	Granulocytic: Gr-1 (Ly-6G) IgG2b, 01211A
  D8 IgG2b, 09821D	Eryth: Ter-119 IgG2b, 09081A

• 
  

• Antibodies for Sorting: (Pharmingen monoclonal rat anti-mouse)

• Sca-1 PE-IgG2a (E31-161.7) 01835B	PE-IgG2a Isotype control, 11025A
  c-Kit (2B8) FITC-IgG2b, 01904D	FITC-IgG2b Isotype control, 11184C
  Fc RII block (clone 2.4G2), 01241D

• 
  

• Mice: 

• C57 (Ly 5.2) 

• C57/SJL (Ly 5.1, Pep3b)

•    

• Other Reagents:

• Dynabeads (Sheep anti-rat, Dynal #110.08)

• Ficoll

• CH296

• Methylcellulose (Fisher)

• Deionized BSA (Sigma)

• Protomine Sulfate

• 
  

• Cytokines: (Peprotech)

• mu SCF

• hu IL-6

• hu Flt-3L

• hu IL-11

• Thrombopoeitin

• hu IL-3

• Erythropoeitin (Amgen)

PROCEDURE

Harvest Marrow:
1. Flush femurs with PBS/FBS and pass cells through a 23G needle.
2. Also collect T-cells as a flow cytometry control.
3. Collect low density cells by equilibrium centrifugation over Ficoll-Hypaque (d=1.077 g/mL).
    Alternatively, RBC may be lysed.  See blood flow cytometry protocol for details.
4. Wash cells in PBS/FBS.

Lineage Depletion: 
1. Resuspend at a density of 5x10⁷ cells/mL in PBS/FBS plus antibody cocktail (each antibody final dilution = 1/500 v/v).
2. Incubate on ice for 30 min. Meanwhile, wash Dynabeads 2x in PBS/FBS.  Resuspend the beads at their original concentration (4 x 10⁸ beads/mL).
3. Wash cells in PBS/FBS, spin, and resuspend at 10⁸ cells/mL. 
4. Slowly add 1 vol. of Dynabeads (4 beads/cell) to the cell suspension. Incubate 5 min. @ room temp. 
5. Expose to magnetic field in a 3 mL round bottom tube or eppendorf tube with the correct sized magnet. Transfer non-adherent cells to a new tube. 
6. Remove tube with beads from magnet.  Add 1/2 vol. PBS/FBS, gently mix.  Expose to magnet again and pool non-adherent cells with the first.
7. Optional:  Repeat the lineage depletion with a new round of antibody and beads (steps 1 - 5).
8. After the last mangetic bead depletion expose the cells to the magnet a second time to remove any remaining beads.  Again, save only the non-adherent cells. 

Cell Sorting (Lin-,cKit+,Sca1+): 
1. Incubate cells in FcgRII block on ice for 10 min. 
2. Stain an aliquot and with PE-IgG2a and FITC-IgG2b isotype antibodies as neg. controls. Incubate remainder of cells with Sca-1 PE, plus c-kit FITC antibodies on ice for 30 min (all antibodies 1/100 v/v).
4.  Wash with 10 mL of PBS/FBS, spin and resuspend in PBS/FBS with 1 µg/mL of propidium iodide. Filter through 70 µm nylon mesh. 
5. Select cells which are PI negative, c-kit positive, Sca-1 positive with intermediate forward and side-scatter. (Run T-cells as a size control).

Viral Transduction: 
1. Pre-coat 12 well plates with CH-296. To each well add 1.5 mL of Iscove's/20% FBS plus growth factors (SCF, IL-6, Flt-3L, IL-11). 
2. Collect 5000 cells per well into 12 well plates with 1.5 mL/well of Iscove's transduction media. 
3. Incubate 48 hrs at 37°C, 5% CO_². 
4. Harvest cells by spinning at 1000 rpm for 5 min. 
5. Add 0.5 mL of 3x growth factors to each well (depending on the experiment). 
6. Resuspend cells in 1 mL of viral supernatant (or control media) and return to wells. 
7. Incubate 12 hrs at 37°C. 
8. Repeat viral transduction (steps 4 to 7). Incubate another 12 hrs at 37°C. 
9. Harvest cells and replate in 1.5 mL of Iscove's/FBS plus growth factors (no virus). 
10. Incubate 4 days at 37°C.

Transplant:
1. Harvest fresh competing marrow from a C57/SJL (Ly 5.1) mouse. Pass through 23G needle and resuspend in Iscove's/FBS at 10⁶ cells mL. 
2. Harvest cells, wash with Iscove's/FBS. 
3. Spin at 1400 rpm for 5 min. and resuspend in 1.5 mL. Record cell count. Add 1.1 mL of cells to 1.1 mL of competing marrow cells. Transplant into lethally irradiated (10 Gy, ⁶⁰Co) recipients. 
4. Of remaining cells save some for FACS analysis for GFP + lineage specific antibodies; the remainder can be used for CFU assays in methylcellulose. 

CFU assays:
1. For CFU assays: Plate an average 20 cells in 35 mm dishes with 1 mL aMEM and 1.2% methylcellulose plus growth factors. 2. Incubate at 37°C in 5%CO2 (plus 5% O2 if available).
3. Analyze for erythroid colonies at 8 days (and confirm by Benzidine staining). 
4. At 12 days analyze for granulocyte/macrophage colonies and large (>1.5mm) mixed colonies (erythroid, meg, and granulocytes confirmed by Giemsa staining). 

GFP and cell surface marker immunostaining: 
1. Incubate cells with lineage specific antibodies as described for cell sorting, above. If live cells are not needed fixation with dilute formalin or paraformaldehyde is O.K. but alcohol based fixatives should be avoided because they allow GFP to leak out of cells. 
2. For unconjugated antibodies, incubate with an anti-IgG2 PE-conjugated secondary antibody. 
3. Wash and incubate with P.I. as for sorting. Analyze on fluorescent microscope or FACS machine.