Therefore, the rationale for our approach is to develop a gentle approach for cell isolation. Systems are needed that can rapidly and efficiently isolate specific cell types without disrupting cell-surface receptor-levels in order to improve cell isolation and enrichment for biomedical applications. These large time scales can affect physiological response and expression levels, resulting in analyses that are not representative of the physiological response 3.
#ITCN IMAGEJ VERIFICATION#
Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.Ĭurrent bench-top cell separation approaches ( e.g., fluorescence activated cell sorting 1, laser capture micro-dissection 2, immuno-magnetic bead separation 1) can take several hours of preparation and sorting. Applications of this technology to xenografts and cancer separation studies are investigated. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. This reduction in harsh reagents and shear forces reduces changes in cellular expression. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin.
In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient.
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