Isolation and Enrichment of Murine Bone Marrow Cells

Mice were handled and euthanized by cervical dislocation according
to guidelines and animal protocols approved by the German authorities.
Using forceps and scissors, the animal’s legs and spines were
dissected. To isolate femurs, tibiae, iliae, and vertebrae, connective
tissue was removed with a scalpel. The isolated bones were then kept
on ice in PBS (Sigma, Cat# D8537)-filled 6-well plates until all bones
were collected.
Note that cells must be kept on ice from the
moment the bones are isolated from the mouse. Bones were gently crushed
twice with 5 mL of ice-cold PBSs using a mortar and pestle until they
turned completely white. The cell suspension was then filtered through
a 40 μm sterile filter (Corning, Cat# 352340) into a 50 mL falcon
tube and kept on ice.
Next, the lysis of erythrocytes was performed.
Briefly, the cell
suspension was centrifuged at 400g for 5 min at 4
°C, followed by the removal of the supernatant. The pellet was
then resuspended into 2 mL of ice-cold ACK lysis buffer (Lonza, Cat#
10-548E) and incubated on ice for 5 min (incubation time may be increased
up to 10 min depending on the size of the pellet). The reaction was
then stopped by adding 10 mL of ice-cold PBS. Then, the cells were
centrifuged at 400g for 5 min at 4 °C, and the
supernatant was discarded.
Lineage negative (Lin−) cells
were enriched using the Dynabeads
Untouched Mouse CD4 Cells Kit (Invitrogen, Cat# 11415D). Briefly,
cells were resuspended in a 500 μL of lineage cocktail (100
μL of the cocktail provided in the kit plus 400 μL of
PBS) and transferred into a 2 mL tube. The incubation step was performed
for 35 min on a rotating wheel at 4 °C. Meanwhile, 400 μL
of Dynabeads were washed twice with 1 mL of PBS on a magnet and resuspended
in 500 μL of PBS.
Next, the cells were washed with 12
mL of ice-cold PBS in a 15
mL falcon tube and centrifuged at 400g for 5 min
at 4 °C. After the removal of the supernatant, cells were resuspended
in 1 mL of ice-cold PBS and incubated together with the prepared Dynabeads
for 20 min on a rotating wheel at 4 °C.
For depletion of
lineage-positive cells, the suspension was incubated
5 min on ice on the depletion magnet until the solution cleared. The
entire supernatant was then transferred into a new FACS tube and kept
on ice. Then, 1 mL of ice-cold PBS was added to the beads and mixed
by vortexing. The depletion step was then repeated, and the two supernatants
combined. Then, the cell suspension was centrifuged at 400g for 5 min at 4 °C, and the supernatant was removed.