eBook: Breakthroughs and Innovations in Immunology Research - 15

a primary lymphoid tissue, responsible for the production
of erythrocytes, granulocytes, monocytes,
lymphocytes and platelets.6,7
Despite its importance
in blood and other disorders, its complexity
has made it difficult to fully characterize all the cell
precursors and their physiological functions.
Our aim with this study was to examine a relatively
unknown precursor, characterized by expression
of CX3CR1, and its capacity to generate cells that
could display either macrophage or dendritic cell
characteristics, depending on the culturing conditions
induced by GM-CSF alone, or GM-CSF and IL4.
However, initial experiments soon revealed the
challenges of isolating such population from bone
marrow, as there were large proportions of unrelated
cells that could potentially reduce the number
of CX3CR1+
cells to be selected by the magnetic isolation.
A particularly large proportion of these unwanted
cells were identified as granulocytic cells,
defined by high expression of Ly-6G (Figure 1).
Thus, we designed an isolation strategy to first
deplete these granulocytic cells, using Ly-6G conjugated
magnetic nanoparticles. This would maximize
the chance to obtain higher purity, while
maintaining an acceptable yield when trying to
perform positive selection of CX3CR1+
cells. The
positive selection would be performed using CX3CR1
biotinylated antibodies, followed by magnetic
Streptavidin nanoparticles.
The complete experimental design, including the
subsequent cell culturing in 24-well plates in the
presence of the different cytokine supplements is
shown in Figure 2.
Following this strategy, we were able to enrich the
CX3CR1 positive fraction more than 10 fold. Working
with low frequency cells is always a challenge,
as the number of positive events is scarce and cell
loss is difficult to overcome. However, we were able
to optimize the protocols and reagents used, and
found a robust strategy that worked consistently
even for such a rare cell population that does not
surpass 7% of total cells in murine bone marrow
(Figure 3).
Figure 1. The percentage of Ly-6G+
cells in mouse bone
Figure 2. Experimental layout to isolate CX3CR1+ cells, differentiate
marrow can be relatively large; it may be advantageous
to deplete this population before enriching CX3CR1+
cells.
them to dendritic cells or macrophages, and adoptively transfer
them to a recipient mouse and monitor cell migration.
15
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