A quantitative image cytometry technique for time series or population analyses of signaling networks.
BACKGROUND
Modeling of cellular functions on the basis of experimental observation is increasingly common in the field of cellular signaling. However, such modeling requires a large amount of quantitative data of signaling events with high spatio-temporal resolution. A novel technique which allows us to obtain such data is needed for systems biology of cellular signaling.


RESULTS
We developed a fully automatable assay technique, termed quantitative image cytometry (QIC), which integrates a quantitative immunostaining technique and a high precision image-processing algorithm for cell identification. With the aid of an automated sample preparation system, this device can quantify protein expression, phosphorylation and localization with subcellular resolution at one-minute intervals. The signaling activities quantified by the assay system showed good correlation with, as well as comparable reproducibility to, western blot analysis. Taking advantage of the high spatio-temporal resolution, we investigated the signaling dynamics of the ERK pathway in PC12 cells.


CONCLUSIONS
The QIC technique appears as a highly quantitative and versatile technique, which can be a convenient replacement for the most conventional techniques including western blot, flow cytometry and live cell imaging. Thus, the QIC technique can be a powerful tool for investigating the systems biology of cellular signaling.
Ozaki
Yu-ichi
Y
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Uda
Shinsuke
S
Saito
Takeshi H
TH
Chung
Jaehoon
J
Kubota
Hiroyuki
H
Kuroda
Shinya
S
D000465:Algorithms; D000818:Animals; D001331:Automation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000032:analysis; D019044:Image Cytometry / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D016716:PC12 Cells; D051381:Rats; D015398:Signal Transduction; D049490:Systems Biology / Q000379:methods*; D013997:Time Factors
D048049:Extracellular Signal-Regulated MAP Kinases