Page 77 - Live-cellanalysis handbook

Live-cell Immunocytochemistry

Coupling protein dynamics to morphological changes
Protein dynamics can also be coupled to morphological changes derivative to induce differentiation, there was a clear association
using IncuCyte® live-cell analysis. Here, measurements of cell between the change in neural cell adhesion molecules (NCAMs)
surface markers were linked to morphological changes in human observed through live-cell ICC and the increase in neurite length
neuroblastoma cells (Figure 3). Here, once treated with a vitamin A over time.

A
Day 0 Day 2

Day 7 Day 14

B

Figure 3: Dynamics of neurite outgrowth and CD56 expression in atRA-differentiated
SH-SY-5Y cells. Cells were incubated with FabFlour-488 conjugated anti-CD56 and treated
with atRA (50μM) at t=0. Blended images show green fluorescence (CD56) and phase
morphology. Neurites are masked in yellow (IncuCyte Neurotrack). The time-course of
upregulation of CD56 (mean values ± SEM, 4 wells) and the relationship between CD56 and
neurite outgrowth (normalized to confluency) are shown in panels A and B, respectively.
In panel B, each symbol represents data taken at different time-points throughout the
experiment. Note the time-dependent increase in neurite length and associated CD56 signal.

Coupling protein dynamics to cell function

Live-cell ICC can also be used to correlate changes in morphology ‘macrophage-like’ cells. To correlate these observations with
with function. In the below example, the differentiation of human function, the ability of differentiated THP-cells to phagocytose
monocytic cells into macrophage-like cells was tracked by IncuCyte® IncuCyte® pHrodo-labeled apoptotic T cells was measured. Only
FabFluor-488 labeling of immune cell surface markers (Figure 4). It those cells treated with PMA were phagocytic, illustrating that it
was found that only phorbol myristate acetate (PMA) produced a is possible to associate protein measurements to the functional
marked change in morphology, yielding large, flattened, adherent properties of cells.

75