Page 125 - Live-cellanalysis handbook

Kinetic Neuroimmune Assays

Differential effects of inhibitors on efferocytosis and phagocytosis
The IncuCyte phagocytosis assays can also be used to evaluate pHrodo labelled E. coli bioparticles. Cytochalasin D is an inhibitor
pharmacological effects on microglial engulfment of dead or of actin polymerization and elicited a concentration-dependent
dying cells as well as bacterial particles. By looking at different inhibition of both efferocytosis and phagocytosis. Cilengitide
types of target material, we can understand how treatments is an inhibitor of αVβ3 and αVβ5 integrins, and it selectively
impact different phagocytic processes, as the receptors that attenuated the efferocytosis of N2A cells with minimal effect
mediate efferocytosis (uptake of dying/dead cells) are different on the phagocytosis of bioparticles. These data support the role
from those that mediate phagocytosis of bacteria and other of αVβ3 and αVβ5 integrins in the cell interactions required for
cellular debris. The BV2 microglia cell line was used to evaluate efferocytosis, but not in the phagocytosis of bacteria-based
the effect of cytochalasin D and cilengitide on both the bioparticles.
efferocytosis of apoptotic N2A cells and the phagocytosis of

Cytochalasin D
Efferocytosis Phagocytosis
5
5
Orange area x 10 Orange area x 10
2
2
(μm /image) (μm /image) Efferocytosis Phagocytosis
1.5 10 µM 0.12 µM No treatment 8 30 µM 0.37 µM No treatment 1.2 6
3.3 µM 0.04 µM No target cells 10 µM 0.12 µM No target cells
1.1 µM 0.01 µM 6 3.3 µM 0.04 µM
1.0 0.37 µM 0.005 µM 1.1 µM 0.01 µM 0.8 4
4
0.5 0.4 2
2

0.0 0 0.0 0
0 6 12 18 24 0 6 12 18 24 -8 -7 -6 -5 -4 -3
Time (h) Time (h) log [Cilengitide] (M)

Cilengitide
Efferocytosis Phagocytosis
5
5
Orange area x 10 Orange area x 10
(μm /image) (μm /image) Efferocytosis Phagocytosis
2
2
1.5 100 µM 1.23 µM No treatment 8 100 µM 1.23 µM No treatment 1.2 6
33 µM 0.41 µM No target cells 33 µM 0.41 µM No target cells
11 µM 0.14 µM 6 11 µM 0.14 µM
1.0 3.7 µM 0.05 µM 3.7 µM 0.05 µM 0.8 4
4
0.5 0.4 2
2

0.0 0 0.0 0
0 6 12 18 24 0 6 12 18 24 -8 -7 -6 -5 -4 -3
Time (h) Time (h) log [Cilengitide] (M)
Figure 5. Quantitative pharmacological analysis of relevant target material. BV-2 effector cells (20,000 cells/well) efferocytose apoptotic N2A cells
(left column) or E. coli bioparticles (middle column). Cytochalsin D (top row) elicits a concentration-dependent inhibition of both efferocytosis and
phagocytosis, yielding IC50 values of 0.16 μM and 1.5 μM, respectively. Cilengitide, and inhibitor of αVβ3 and αVβ5 integrins, selectively attenuates
efferocytosis (IC50 value of 0.16 μM), with minimal effect on phagocytosis at the highest concentration tested (100 μM).

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