Abstract
Purpose::
Develop a high throughput screening (HTS) approach for rapid measure of protein expression levels by primary quantitative immunofluorescence in fixed cells. Elucidating lead candidates for post transcriptional gene silencing (PTGS) requires examination of many species. Slow, multi-step assays (e.g. Western blots) are typically used to evaluate proteins expressed in transfected cells. A method to rapidly quantify a targeted protein would facilitate PTGS development.
Methods::
A Nikon TE-300 inverted microscope equipped with low power lenses and a cooled monochromatic 12-bit CCD camera images an entire well in a 96-well plate. The stage is driven by microstepping X and Y motors, controlled by IP Labs software; Z stage (focus) is controlled by a motor and a piezo sensor element. The system scans the 96-well array in predetermined routes, selecting a focused image from a Z-stack acquisition. A purified monoclonal antibody (Ab) to the protein of interest is labeled with a bright, stable far-red fluorescent dye (e.g. Alexa647), to avoid the bulk of cellular auto-fluorescence. HEK cells grown on optically flat polystyrene surfaces in black-walled 96-well arrays were fixed and permeabilized, and incubated with the far-red exciting primary Ab and a DNA binding blue-exciting fluor. In each well the far-red protein fluorescence is quantified and the uniform nuclear green fluorescence is taken as a measure proportional to cell number.
Results::
Alexa647 labeled mouse monoclonal (1D4) to human rod opsin (Rho) was prepared in uniform batches to consistently achieve several mol/mol of Alexa647/mole 1D4. Naïve HEK293S cells and those stably expressing Rho were prepared and incubated with varying amounts of Alexa647-1D4 Ab. Signal-to-noise levels at different Ab levels and with different objective lenses demonstrated optimized conditions for reliable detection of Rho in fixed cells. Signal levels are proportional to the number of bound far red fluor molecules in any single well. SYTOX Green nuclear labeling provides a quantitative measure proportional to cell number making it a useful assay for ratiometric normalization.
Conclusions::
A microscope based HTP system to quantitate Rho has been demonstrated in proof-of-principle. This system is versatile and can measure any cellular protein provided that Abs are available. Cellular auto-fluorescence is essentially obviated through choice of far-red fluors. Multiple proteins could be screened in a single high content assay. This emerging tool has substantial versatility to rapidly screen agents of gene therapy with robust quantitative resolve.
Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • microscopy: light/fluorescence/immunohistochemistry