Novel full-spectral flow cytometry with multiple spectrally-adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement.

Flow cytometric analysis with multicolor fluoroprobes is an essential method for detecting biological signatures of cells. Here, we present a new full-spectral flow cytometer (spectral-FCM). Unlike conventional flow cytometer, this spectral-FCM acquires the emitted fluorescence for all probes across the full-spectrum from each cell with 32 channels sequential PMT unit after dispersion with prism, and extracts the signals of each fluoroprobe based on the spectral shape of each fluoroprobe using unique algorithm in high speed, high sensitive, accurate, automatic and real-time. The spectral-FCM detects the continuous changes in emission spectra from green to red of the photoconvertible protein, KikGR with high-spectral resolution and separates spectrally-adjacent fluoroprobes, such as FITC (Emission peak (Em) 519 nm) and EGFP (Em 507 nm). Moreover, the spectral-FCM can measure and subtract autofluorescence of each cell providing increased signal-to-noise ratios and improved resolution of dim samples, which leads to a transformative technology for investigation of single cell state and function. These advances make it possible to perform 11-color fluorescence analysis to visualize movement of multilinage immune cells by using KikGR-expressing mice. Thus, the novel spectral flow cytometry improves the combinational use of spectrally-adjacent various FPs and multicolor fluorochromes in metabolically active cell for the investigation of not only the immune system but also other research and clinical fields of use.

Rapid development of hydrophilicity and protein adsorption resistance by polymer surfaces bearing phosphorylcholine and naphthalene groups.

In order to provide a protein adsorption resistant surface even when the surface was in contact with a protein solution under completely dry conditions, a new phospholipid copolymer, poly (2-methacryloyloxyethyl phosphorylcholine (MPC)- co-2-vinylnaphthalene (vN)) (PMvN), was synthesized. Poly(ethylene terephthalate) (PET) could be readily coated with PMvN by a solvent evaporation method. Dynamic contact angle measurements with water revealed that the surface was wetted very rapidly and had strong hydrophilic characteristics; moreover, molecular mobility at the surface was extremely low. When the surface came in contact with a plasma protein solution containing bovine serum albumin (BSA), the amounts of the plasma protein adsorbed on the dry surface coated with PMvN and that adsorbed on a dry surface coated with poly(MPC-co-n-butyl methacrylate) (PMB) were compared. Substantially lower protein adsorption was observed with PMvN coating. This is due to the rapid hydration behavior of PMvN. We concluded that PMvN can be used as a functional coating material for medical devices without any wetting pretreatment.