Tools for quantitative and validated measurements of cells.
In this chapter, we describe the preparation of thin films of collagen that can serve as reference materials for assuring reproducible and predictable cell responses. Subtle differences in the molecular-scale characteristics of extracellular matrix proteins, including the supramolecular structure of type 1 collagen, can have tremendous influences on cell state and cell-signaling pathways; therefore the careful control and analysis of the culture surface is critical to assure a relevant and consistent response in cell-based assays. We also describe how cell-phenotypic parameters such as morphology, proliferation, and green fluorescent protein expression can be unambiguously quantified in adherent cells by automated fluorescence microscopy or high content screening. Careful consideration of protocols, and the use of fluorescent reference materials, are essential to assure day-to-day and instrument-to-instrument interoperability. The ability to collect quantitative data on large numbers of cells in homogeneous matrix environments allows assessment of the range of phenotypes that are reproducibly expressed in clonal cell populations. The inherent distribution of responses in a cell population will determine how many cells must be measured to reach an accurate determination of cellular response.
Plant
Anne L
AL
National Institute of Standards and Technology, Gaithersburg, MD, USA.
Elliott
John T
JT
Tona
Alessandro
A
McDaniel
Dennis
D
Langenbach
Kurt J
KJ
D000818:Animals; D001695:Biology / Q000379:methods*; D002468:Cell Physiological Phenomena; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism; D051381:Rats; D015203:Reproducibility of Results; D013194:Staining and Labeling; D016707:Tissue Fixation
D024042:Collagen Type I; D049452:Green Fluorescent Proteins