Page 124 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

Specific detection and visualization of cell engulfment by microglia
Another important function of microglia cells is the phagocytosis (Figure 3). As the engulfed cell enters the acidic phagosome of
of cellular debris and dead neurons. To visualize and quantify the the microglia, the change in pH results in an orange fluorescent
phagocytic potential of microglia, a non-perturbing pH sensitive signal which is automatically segmented and quantified by
pHrodo dye IncuCyte pHrodo Orange, was used. Neuro-2a integrated IncuCyte software. To verify sensitivity of the assay,
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target cells were treated with staurosporine to induce apoptosis increasing densities of pHrodo labelled apoptotic N2A cells
and subsequently labelled with the pHrodo Orange Cell Labeling were added to pre-plated rat primary microglia. Data reveals
Kit. The target cells were added to wells containing iPSC-derived a N2A density-dependent response of orange intensity due to
microglia and imaged in real time real time to visualize and phagocytosis by microglia, and no fluorescent signal in wells
quantify the engulfment of the labeled apoptotic N2A cells containing only apoptotic N2A cells. (Figure 4).

Figure 3. HD phase and fluorescent visualization of N2a engulfment
by microglia. Time-lapse visualization of iPSC-derived microglia (Axol
BioSciences) engulfing IncuCyte pHrodo Orange-labeled apoptotic
N2A cells. Images verify the entry of apoptotic target cells into the
phagosome of microglia.

Apoptotic N2A alone Microglia with N2A

Fluorescent image Fluorescent image with mask

Orange area x 10
5
Figure 4. Visualization and (μm /image)
2
quantification of apoptotic
N2A engulfment by microglia. 1.0
N2A cells were pre-treated with 50 K/well
staurosporine (24 hrs), labeled 0.8
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with the IncuCyte pHrodo ® 25 K/well
Orange Cell Labeling Kit, and
added to pre-plated primary 0.6
rat microglia (Brain Bits, 20,000
cells/well). N2A cells alone
have minimal fluorescence (left 0.4
image). Engulfment of labeled 12 K/well
apoptotic N2A cells by microglia 0.2
causes and increase in orange 6 K/well
fluorescence (fluorescent image)
that is automatically segmented to 0.0 Control
quantify N2A density-dependent 0 5 10 15
efferocytosis over time (right). Time (h)

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