Combination of Small Molecule Microarray and Confocal Microscopy Techniques for Live Cell Staining Fluorescent Dye Discovery

Abstract

Abstract:Discovering new fluorochromes is significantly advanced by high-throughput screening (HTS) methods. In the present study a combination of small molecule microarray (SMM) prescreening and confocal laser scanning microscopy (CLSM) was developed in order to discover novel cell staining fluorescent dyes. Compounds with high native fluorescence were selected from a 14,585-member library and further tested on living cells under the microscope. Eleven compartment-specific, cell-permeable (or plasma membrane-targeted) fluorochromes were identified. Their cytotoxicity was tested and found that between 1-10 micromolar range, they were non-toxic even during long-term incubations.

AUTEN-67, an autophagy-enhancing drug candidate with potent antiaging and neuroprotective effects

“…Small molecule microarrays were constructed and prepared for screening as previously described. 57,58 Briefly, for printing with MicroGrid II mechanical microarray microspotter (BioRobotics, Cambridge, UK) chemically activated glass slides (Avicor Ltd., Szeged, Hungary) were used, which contain a treelike, branched dendrimer structure with reactive functional groups at the terminal position. 59 Fluorescent-labeled MTMR14 was prepared with Alexa Fluor 647 carboxylic acid succinimidyl ester (Molecular Probes, A-20106) as previously described.…”

Development of novel dyes and fluorescence-imaging based approaches for cellular localization and dynamics of lipid droplets

“…Bar 10 μm. (Molnár and Kuntam et al, 2013) We have discovered an unusual spectral behavior of the chemical C2 during our microscopy analyses. Gradual relocalization of dye signal from mitochondria to cytoplasm (seen as diffused dye signal all over the cell except in the nucleus) and the nucleolus was observed when labeled cells were repetitively scanned with 633 nm laser at 50% intensity setting as shown in Figure 8.…”

“…Bar 10 μm. (Molnár and Kuntam et al, 2013) We have observed that photoconversion was also inducible by illumination with the rhodamine filter set of the fluorescence microscope (510-550 nm green excitation). To confirm the nucleolar localization of the photoconverted C2 dye (denoted with an asterisk C2*), we have used co-staining on live cells, 1 h after photoconversion with a vital DNA dye Hoechst 33342 which displays a negative staining pattern at the RNA-rich nucleolar regions (arrows in Fig.…”

“…Still, this technique has some drawbacks like difficulties in automated image analysis and higher cost compared to SMMs. Thus, the advantages of these two approaches, SMM and CLSM, can be combined and applied for the discovery of new fluorescent dyes (Molnár and Kuntam et al, 2013). …”

Investigation of the role of prion protein in protection against transition metal induced toxicity

“…The prion protein's role in a particular cellular process, including those that are concerned with metal-PrP interrelations in ex-vivo and in-vivo systems are generally studied by either genetically ablating (Büeler et al, 1992;Haigh and Brown, 2006;Kim et al, 2005;Kuwahara et al, 1999;Rossi et al, 2001;Sakaguchi et al, 1996) or siRNA silencing (Loubet et al, 2012) while working on HeLa cells (Molnár et al, 2013)., and have used here similarly. The bright red immunofluorescence-staining pattern in the ZW 13-2 cells revealed that the prion protein was distributed on the surface of the cells, whereas no immunoreactivity was detected in the Zpl 2-1 cells.…”