Cell monitoring Image analysis

The suppression and recovery of protein synthesis from DTT treatment (without cycloheximide treatment) can be monitored via metabolic pulse radiolabeling of cell cultures using [35S]-methionine and subsequent determination of radiolabeled protein content either by SDS-PAGE/ phosphor-imager analysis or liquid scintillation of tricholoroacetic acid insoluble material (Stephens et al., 2005). 

An analogous procedure based on injection of GFP-transfected cells (see below) and monitoring by image analysis may give information on the pathways and growth of invasive cells. 

Maruhashi, F., Murakami, S., and Baba, K. (1994) Automated monitoring of cell concentration and viability using an image analysis system. Cvtotechnol. 15 281-289... 

A more direct optical method has been introduced whereby a membrane-permeant, charged dye equilibrates across the membrane of interest. Ideally the dye will equilibrate rapidly and show minimal binding to materials on either side of the membrane (34). Using quantitative confocal fluorescence microscopy, the fluorescence of the dye on either side of the membrane is measured as fluorescence intensity is linearly proportional to fluorophore concentration, the measured intensities can be substituted for the ion concentrations ([M" ] and [M ]o) in equation 1 and the potential obtained (34). This technique is ultimately limited by the spatial resolution of the confocal microscope—it works well for large compartments in whole cells. Recent computational approaches to image analysis have allowed the spatial resolution to be increased to the level where mitochondrial membrane potential can be monitored by such means in individual cells (35). 

The extent of cell migration/invasion can be monitored by standard light microscopy and/or by densitometric analysis (e.g., using Image software). Normalize the extent of migration in each sample to the one of the corresponding internal control. 

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