![]() This photo linker can be cleaved by irradiation of 365 nm UV light, which has been proven to be harmless to cells. We have chosen the -butanoic acid derivative 20, 21, 22, 23 as a photo-cleavage linker ( Figure 1b). After the irradiation and release of the peptide, the cytotoxic behavior of the peptide can be monitored. For the toxicity assay, UV light irradiation on the desired wells in a 96-well plate releases the peptide from the plate bottom. Initially, a peptide is immobilized on a commercially available plastic culture plate bottom via a photo-cleavable linker. Consequently, in this study we attempted to construct a peptide photo-release system for cytotoxicity analysis ( Figure 1a). 15 In addition, by applying statistical analyses, such as hierarchical clustering analysis, to cell fingerprints, high-confidence correlations between cell types and biological functions were elucidated based primarily upon peptide charge and hydrophobicity data.Īs is done for the development of protein microarray technology, 6 we are currently focusing on development of a more practical array format 16, 17, 18, 19 and on achievement of a more detailed detection and analysis method for use in cellular research. In our cell array technique, the obtained cellular response patterns upon stimulation with library peptides were regarded as ‘cell fingerprints’, which revealed how peptide properties affect a particular cell type. 10, 11, 12, 13, 14 We then began to explore the designed peptide arrays for use in cell microarrays. From this point of view, we have constructed peptide microarrays composed of various secondary structures, 4, 5, 6, 7, 8, 9 and have initially applied the designed peptide arrays to protein-analysis systems. Cell microarrays provide an attractive solution, as they offer more than a simple miniaturization and mechanization of conventional equipment. 2, 3 To analyze the phenotypic consequences of mammalian cells with chemical stimulants including drugs, systematic cell-based assays in a high-throughput screening format are increasingly indispensable. This study implies that the peptide photo-releasing array system will allow the realization of high-throughput cell arrays for cellomics analyses and cell-based phenotypic drug screenings.Īfter the elucidation of genome sequences in various organisms, significant advances in ‘omics’ studies have opened up cellomics research based on cell-based phenotypic assays 1 for studying vital cellular phenomena. Throughout this study, we successfully immobilized peptides via the photo-cleavable linker, released them by UV irradiation spatiotemporally and conducted the cell-toxicity assay. This relative toxicity nearly corresponded to that of ∼10 μ M TMR-KLA-C in solution, and we found that the released peptide concentration per well was ∼10 μ M at 60 min irradiation. All cell types showed ∼25% lowering of viability with the photo-released 5-(and-6)-carboxytetramethylrhodamine (TMR)-GKLAKLAKKLAKLAKKLAKLAKGC (TMR-KLA-C) peptide compared with the non-coated plate. Cell-toxicity assays with this system for three cell types were performed. The immobilized peptide showed no cytotoxicity therefore, the cells could be cultured on the peptide-immobilizing plate from the beginning of the experiment. Immobilization and light-irradiation conditions were optimized. As a result, the cytotoxic behavior of the peptide could be monitored. Once UV light irradiated the desired wells, the peptide was released from the bottom. In this system, a peptide was immobilized on a conventional 96-well plate bottom via a photo-cleavable linker. We constructed a novel peptide-array format system for cellular toxicity analysis.
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