Page 26 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

To capture the stimulus dependent effect on T-cell proliferation illustrated in Figure 10A, little or no cell proliferation was observed
and cluster formation, isolated human PBMCs were seeded (20 K/ in the presence of IL-2 alone, however, moderate proliferation
well) on a PLO-coated flat bottom 96-well plate and grown in the was observed with the addition of a-CD-3. T-cell proliferation
presence of various T-cell activator combinations, hrIL-2 (10 ng/ was further enhanced by the addition of a-CD-28, revealing a
ml), a-CD-3 (0.1 ng/ml) and/or a-CD28 (1-100 ng/ml). HD phase concentration dependent response (Figure 10A). A similar stimulus
contrast images were captured on the IncuCyte system over 240 dependent cluster formation profile was also measured in the
hours and analyzed for phase confluence (%) as a measure of presence of various T-cell activator combinations (Figure 10B).
cell proliferation and the number of phase objects above an area These data show the potential of this kinetic and morphological
threshold (cluster count/well) to define cluster formation. As approach to evaluating immune cell activation.

A Proliferation
% confluence
% confluence 216 h
100 100
IL-2/anti-CD3 (0.1)/CD28 100ng/mL

IL-2/anti-CD3 (0.1)/CD28 10ng/mL
80 80

IL-2/anti-CD3 (0.1)/CD28 1ng/mL
60 60
IL-2/anti-CD3 0.1 ng/mL

40 40
n=5 replicates

20 20

IL-2 10 ng/mL
0 0
48 96 144 192 240 1 2 3 4 5
Time (h)
B Cluster formation
Cluster count 168 h Cluster count 168 h
(per well) (per well)
1000 1000
IL-2/anti-CD3 IL-2/anti-CD3 (0.1)/CD28 10ng/mL
(0.1)/CD28 100ng/mL
800
750 IL-2/anti-CD3 (0.1)/CD28 1ng/mL

600

500
IL-2/anti-CD3 0.1 ng/mL 400

250
200

0 IL-2 10 ng/mL 0.0
48 96 144 192 240 1 2 3 4 5
Time (h)
Figure 10. T-cell activation is stimulus and concentration-dependent. Data shown are for PBMCs treated with combinations of IL-2, anti-CD3, and anti-CD28.

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