The presence or absence of cellular proteins can indicate a reaction to stimulation, a state of differentiation, unique characteristics of a specific cell type, or success of a gene transfection. For example, the surface expression of Vascular Cell Adhesion Molecule-1 (VCAM-1) is usually absent from normal vascular endothelium but can be stimulated in vitro by the cytokine TNF-α to elicit an inflammatory response. Such protein expressions in a cell can be readily visualized and easily quantified using imaging cytometry.
Quantitative measurement of marker expression in cells
Human umbilical vein endothelial cells (HUVEC) were cultured in 96-well plates for 48 hours and then treated for 2 hours with anti-inflammatory compounds, SB202 and SB203, at varying doses. Following the treatment, VCAM-1 expression was stimulated with TNF-α for 24 hours. Cells were then fixed using 4% paraformaldehyde and stained with anti-VCAM fluorescein-conjugated antibodies. Images were acquired using SpectraMax® MiniMax™ Imaging Cytometer, a field upgrade option for the SpectraMax® i3 Multi-Mode Detection Platform. Image analysis was performed on-the-fly using the Marker Expression Protocol of SoftMax® Pro Software. Compound efficacy was evaluated with dose-response curve in SoftMax Pro Software, showing whether the anti-inflammatory compounds were successful in protecting the cells from cytokine stimulation, thus inhibiting subsequent VCAM-1 expression.
The level of VCAM-1 expression was quantitated by setting an intensity threshold to filter out low background fluorescence and a size threshold to filter out artifacts tiny artifacts such as scratches, dust, or bubbles. Figure 1 shows the visually guided setup of the Marker Expression Protocol in SoftMax Pro Software.
Fixed cells stained with fluoresceinconjugated anti-VCAM are identified with the Marker Expression Protocol. Since cells are nearly confluent, the protocol is set up to measure overall marker expression in the field of view instead of segmenting individual cells.
More information from your marker expression assays
The SpectraMax MiniMax Imaging Cytometer provides visualization and information beyond simple fluorescence intensity in the well. Measured areas of fluorescence are localized to the cells expressing the markers-of-interest and the large imaged field-of-view allows a significant number of cells to be interrogated at once. Intuitive analysis using SoftMax Pro Software allows plotting of dose response curves and calculation of IC50 values. Taken together, the MiniMax Imaging Cytometer simplifies cellular imaging and provides results that complement standard plate reader assays for measuring marker expression in cells.