LINEAGE DEPLETION OF WHOLE BONE MARROW CELLS
Introduction
Hematopoietic stem cells (HSCs) and progenitor cells (such as mesenchymal stem cells/ progenitors)
comprise a small percentage of whole bone marrow. These cells are “lineage-negative”, i.e. do not
express any of the hematopoietic differentiation markers. For methods involving stem and progenitor
cell isolation, it is best to first deplete the bone marrow of lineage-positive cells to enrich for this
immature cell population
Buffers and Materials
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Mouse instruments (forceps, scissors, scalpel)
Mortar and pestle
1X DPBS (Cellgro, 21-031-CV)/ 2% FBS (Hyclone, #SH30070.03)
Pre-titered saturating lineage depletion antibody mix (we use rat anti-mouse TER-119, CD5,
CD4, CD8, B220, CD11b, Gr-1; all purified antibodies obtained from eBioscience)
50 ml conical tubes
Sterile FACS tubes (capped)
40m mesh filters
Ficoll-Hypaque (GE Healthcare, #71-7167-00 AG)
Sheep anti-rat Dynabeads (Dynal, from Invitrogen #110.35)
Magnet particle concentrator (Dynal)
Other antibodies (for CFU-F precursors/MSCs, these are CD45, Sca-1, CD31 in different
color conjugates)
Protocol
1. Obtain femurs, tibiae and iliac crests from the mice. Clean all fat and muscle away using a
scalpel.
2. Crush bones in mortar and pestle with 1X DPBS/2% FBS.
3. Filter into 50 ml conical tubes through 40 m mesh filters.
4. Do cell counts (do this in white cell fluid, which contains 3% acetic acid to hemolyse the
erythrocytes).
5. Centrifuge tubes at 1400 rpm for 5 minutes at 4C.
6. Layer 4 ml cell suspension (in PBS/% FBS) over 3 ml Ficoll gradients (in 15 ml conical
tubes). Centrifuge Ficoll gradients at 1800 rpm for 10 minutes at 4C with break off
(deceleration = 0).
7. Remove majority of top (aqueous) phase with Pasteur pipette and discard
8. Remove interphase (high density cells) into 50 ml conical tubes with excess PBS/2% FBS (to
wash).
9. Take an aliquot of cells to do a cell count.
10. Centrifuge at 1400 rpm for 5 min.
11. Resuspend cells n lineage-depletion antibody mix – 50 l per 5 x 106 cells (lineage antibodies
are 1/500 final each of rat anti-mouse CD4, CD8, CD5, B220, Gr-1. CD11b, TER119 in
PBS/2% FBS). Incubate on ice for 30 min.
12. Wash cells with PBS/2% FBS. centrifuge at 1400 rpm for 5 min.
Updated 1/25/11 by RK
13. Aspirate off supernatant, place cells in 50 ml conical tube at 1x108 cells/ml.
14. Add sheep anti-rat Dynabeads (4x108 beads/ml) at same volume as cell suspension (4 beads
per cell).
15. Centrifuge at 400 rpm for 3 min.
16. Aspirate off supernatant, resuspend in 1 ml PBS/2%FBS in 5 ml FACS tube.
17. Place on magnetic column.
18. Remove non-magnetic cells (these are the ones not bound to the column with the beads
19. An extra step of depletion can be done if ultra-pure cells are needed. In this step, use half the
volume of beads used in the first depletion.
20. Count cells, centrifuge at 1400 rpm for 5 min.
21. These are lineage negative bone marrow cells
22. To stain for the CFU-F progenitors, use the following:
 CD45-PE
 Sca-1-PE-Cy5.5
 CD31-FITC
 Single color controls from one sample are also useful to set up the FACS machine
properly – just small aliquots of these (less than 500,000 cells) are required. An
unstained sample is also useful.
 Stain the cells at 5x106 cells/50 l with 1/500 of the antibodies (30 min at 4C)
 Wash aspirated off supernatant, resuspend in 300 l per tube and analyze on the FACS
machine.
Updated 1/25/11 by RK