Real-time quantification of protein expression and translocation at individual cell resolution using imaging-dish-based live cell array

Abstract

Cell populations represent intrinsically heterogeneous systems with a high level of spatiotemporal complexity. Monitoring and understanding cell-to-cell diversity is essential for the research and application of intra- and interpopulation variations. Optical analysis of live cells is challenging since both adherent and nonadherent cells change their spatial location. However, most currently available single-cell techniques do not facilitate treatment and monitoring of the same live cells over time throughout multistep experiments. An imaging-dish-based live cell array (ID-LCA) has been developed and produced for cell handling, culturing, and imaging of numerous live cells. The dish is composed of an array of pico scale cavities—pico wells (PWs) embossed on its glass bottom. Cells are seeded, cultured, treated, and spatiotemporally measured on the ID-LCA, while each cell or small group of cells are locally constrained in the PWs. Finally, predefined cells can be retrieved for further evaluation. Various types of ID-LCAs were used in this proof-of-principle work, to demonstrate on-ID-LCA transfection of fluorescently tagged chimeric proteins, as well as the detection and kinetic analysis of their induced translocation. High variability was evident within cell populations with regard to protein expression levels as well as the extent and dynamics of protein redistribution. The association of these parameters with cell morphology and functional parameters was examined. Both the new methodology and the device facilitate research of the translocation process at individual cell resolution within large populations and thus, can potentially be used in high-throughput fashion.

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Abbreviations

3D:

Three dimension

BF:

Bright field

CV:

Coefficient of variation

DRIE:

Deep reactive ion etching

FC:

Flow cytometry

FDA:

Fluorescein diacetate

FI:

Fluorescence intensity

HTS:

High-throughput screening

ID-LCA:

Imaging-dish-based live cell array

MC:

Microcavity

NPs:

Nanoparticles

PCR:

Polymerase chain reaction

PDMS:

Polydimethylsiloxane

PWs:

Picowells

ROI:

Region of interest

SD:

Standard deviation

TL:

Translocation level

UV:

Ultraviolet

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Acknowledgments

This study was endowed by the Bequest of Moshe-Shimon and Judith Weisbrodt. EGFP-PKCδ vector was a generous gift from Prof. Chaya Brodie's laboratory at Bar-Ilan University.

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Correspondence to Mordechai Deutsch.

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Ravid-Hermesh, O., Zurgil, N., Shafran, Y. et al. Real-time quantification of protein expression and translocation at individual cell resolution using imaging-dish-based live cell array. Anal Bioanal Chem 406, 7085–7101 (2014). https://doi.org/10.1007/s00216-014-8157-1

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Keywords

  • Fluorescence
  • Microfabrication
  • Biochip
  • Single-cell analysis
  • Imaging
  • Kinetics