Epigenetic Analysis After Single Cell Interactions Using Microfluidics
AssigneeNortheastern University
InventorsTania KONRY, Agustin DE GANZO, Judene A. THOMAS, Christina SHARKEY
Life of this patent
Estimating the term…
Abstract
Single-cell methods are provided for investigating DNA and RNA interactions with regulatory proteins and evaluating the cytotoxicity of immune cells towards target cells, such as tumor cells. The methods utilize droplet microfluidics and imaging-based technology to facilitate the isolation and analysis of protein-bound nucleic acids from individual immune cells or apoptotic tumor cells.
Claims29
A method for epigenetic analysis of one or more cytotoxic immune cells, the method comprising the steps of:
- (a) providing a first aqueous suspension comprising cytotoxic immune cells, a second aqueous suspension comprising target cells, and a microfluidic device configured for co-encapsulating and imaging individual said cytotoxic immune cells with individual said target cells in aqueous microdroplets in an oil stream;
- (b) forming a plurality of said aqueous microdroplets, distributing and imaging the microdroplets in a plurality of docking stations in said microfluidic device, and incubating and imaging the microdroplets for a period of time;
- (c) selecting a portion of the microdroplets in which killing of a said target cell has occurred according to selected criteria;
- (d) collecting and merging the selected microdroplets;
- (e) fixing the contents of the merged microdroplets with a chemical fixing agent, whereby cytotoxic immune cells are fixed;
- (f) lysing the fixed cells and isolating nuclei of the fixed cytotoxic immune cells;
- (g) immobilizing the isolated nuclei on a solid support;
- (h) incubating the immobilized nuclei with an antibody that specifically binds to a selected nucleic acid-binding protein;
- (i) incubating the immobilized nuclei with a nuclease, whereby nucleic acid not bound to the selected nucleic acid-binding protein is degraded, leaving nucleic acid fragments that were bound to the selected nucleic acid-binding protein;
- (j) isolating said nucleic acid fragments;
- and
- (k) sequencing one or more of the isolated nucleic acid fragments, whereby one or more nucleic acids interacting with the selected nucleic acid-binding protein in said one or more cytotoxic immune cells are identified.
(canceled)
A method of epigenetic analysis of one or more target cells following interaction between said one or more target cells and one or more cytotoxic immune cells, the method comprising:
- (a) providing a first aqueous suspension comprising cytotoxic immune cells, a second aqueous suspension comprising target cells and microbeads coated with a capture agent for a selected target cell nucleic acid-binding protein, and a microfluidic device configured for co-encapsulating and imaging individual said cytotoxic immune cells with individual said target cells in aqueous microdroplets in an oil stream;
- (b) forming a plurality of said aqueous microdroplets, distributing and imaging the microdroplets in a plurality of docking stations in said microfluidic device, and incubating and imaging the microdroplets for a period of time, whereby in a portion of the microdroplets a said target cell has been killed by a said cytotoxic immune cell, and whereby the microbeads bind to the nucleic acid-binding protein in the killed target cells;
- (c) selecting a portion of the microdroplets in which killing of a said target cell has occurred according to selected criteria;
- (d) collecting and merging the selected microdroplets;
- (e) isolating said microbeads;
- (f) releasing from the isolated microbeads nucleic acid fragments that were bound to said nucleic acid-binding protein;
- (g) isolating said nucleic acid fragments;
- and
- (h) sequencing one or more of the isolated nucleic acid fragments, whereby one or more nucleic acids interacting with the nucleic acid-binding protein in said one or more target cells are identified.
(canceled)
A kit for epigenetic analysis of a cytotoxic immune cell, the kit comprising:
- (i) a microfluidic device configured for co-encapsulating and imaging individual said cytotoxic immune cells with individual said target cells in aqueous microdroplets in an oil stream;
- and
- (ii) an antibody that specifically binds a selected nucleic acid binding protein of the cytotoxic immune cell.
(canceled)
A kit for epigenetic analysis of a target cell, the kit comprising:
- (i) a microfluidic device configured for co-encapsulating and imaging individual said cytotoxic immune cells with individual said target cells in aqueous microdroplets in an oil stream;
- and
- (ii) a plurality of microbeads comprising a capture agent, such as an antibody or aptamer, that specifically binds a selected nucleic acid binding protein of the target cell.
(canceled)
Dependencies are inferred from the claim text.