MEASURING FREQUENCY OF PATHOGEN-SPECIFIC T CELLS IN PERIPHERAL BLOOD AS ESTABLISHED BY TCR-INDUCED CA(2+) SIGNALING

Abstract: a method for measuring kinetics of caflux in differentially responding t cells that form monolayer on the glass surface in response to antigenic peptides or live target cells comprising: immobilizing t cells labeled with casensitive fluorophore on the glass bottom of a well, covered with capturing antibody or a capturing protein that bind to non-stimulatory t-cell surface receptor; adding to the well a single or multiple peptide epitopes that binds to the cell surface mhc molecules to be presented for recognition by cognate t cells; the stimulatory signal could also be delivered by live target cells that display peptide epitope(s); wherein the recognition of stimulatory of pmhc by the peptide specific t cells leads to increase of intracellular calevel and fluorescence intensity in the responding t cells, which is then identified after the subtracting fluorescence intensity for every t cell before and after the addition of the peptide antigens; scoring each responding t cell into a category according to three categories including: a rapid and sustained t-cell response, an oscillatory response, or a delayed and oscillatory response; and measuring changes in number of an individual t cells with increased intracellular fluorescence as function of time provides the kinetic curve of the tcr-mediated ca signaling.

Inventor(s): Yuri Sykulev, Nadezhda Anikeyeva

CPC Classification: G01N33/582 ({with fluorescent label})

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