Abstract: Disclosed herein are methods for detecting a target protein in a sample and methods for detecting a target protein and a target nucleic acid in a sample. Kits for carrying out the methods are also disclosed.

Abstract: Provided herein are compositions, systems, kits, and methods for detecting the presence or absence of a target protein in a discrete entity comprising: a) generating a discrete entity (e.g., microdroplet) comprising: i) a first cell that may secrete, or surface express, a target protein, ii) a quenched oligonucleotide probe, iii) first antibody-oligonucleotide conjugate or a particle-oligonucleotide conjugate, and a second antibody-oligonucleotide conjugate that bind the target protein in proximity to each to form an oligonucleotide template structure (OTS), and a nickase enzyme that cleaves the quenched oligonucleotide probe when it is hybridized to the OTS such that a detectable dye (e.g., fluorescent dye) is released and generates a signal; and b) detecting the presence or absence of the signal from the detectable dye.

Abstract: The present invention provides compositions, systems, kits, and methods for combining a. single myeloma cell and a single B-cell (e.g., from an animal exposed to a desired antigen) via discrete entity (e.g., droplet) microfluidics. In certain embodiments, a microfluidic device is used to merge a discrete entity containing a B-cell, and a discrete entity containing a myeloma cell, and a discrete entity containing gellable material, at a merger region via a trapping element in order to generate a combined discrete entity. In further embodiments, the combined discrete entity is treated such that a gelled discrete entity is formed.

Abstract: The present disclosure provides methods, compositions, and systems employing blocked primers. Aspects of the disclosure include providing a blocked primer reaction mixture that includes a blocked primer and a template nucleic acid component from a single cell; unblocking the blocked primer to produce an active primer reaction mixture and subjecting the activated primer reaction mixture to primer extension conditions, such as nucleic acid implication conditions.

Abstract: The present invention provides compositions, systems, kits, and methods for combining a. single myeloma cell and a single B-cell (e.g., from an animal exposed to a desired antigen) via discrete entity (e.g., droplet) microfluidics. In certain embodiments, a microfluidic device is used to merge a discrete entity containing a B-cell, and a discrete entity containing a myeloma cell, and a discrete entity containing gellable material, at a merger region via a trapping element in order to generate a combined discrete entity. In further embodiments, the combined discrete entity is treated such that a gelled discrete entity is formed.

Abstract: The present invention provides compositions, systems, kits, and methods for analyzing the interaction of T-cells and neoantigen presenting cells (and other cells) via discrete entity (e.g., droplet) microfluids. In certain embodiments, a microfluidic device is used to merge a discrete entity containing a T-cell, and a discrete entity containing a neoantigen presenting cell, at a merger region via a trapping element in order to generate a combined discrete entity. In particular embodiments, at least one thousand of such combined discrete entities are formed in about one second. In some embodiments, whether the receptor on the T-cell sufficiently binds the neoantigen to activate the T-Cell is detected (e.g., via detection of cytokine or granzyme B release). In certain embodiments, provided herein are methods for identifying polyfunctional T-cells or NK-cells, as well as methods of screening for such cells that would be cytotoxic if injected into a subject.

Abstract: Disclosed are compositions and methods for the multiplexed analysis of one or more intracellular targets of a single cell. Exemplary compositions of the disclosure comprise a surface comprising a plurality of capture agents operatively-linked thereto, wherein each capture agent specifically binds to a distinct intracellular target and wherein the plurality of capture agents form a repeating pattern; a substrate comprising a plurality of chambers, wherein the substrate releasably couples with the surface and wherein each chamber of the plurality of chambers comprises at least one repeat of the repeating pattern of the plurality of capture agents of the surface; a coating composition comprising a cell lysis composition; and a linker composition comprising a functionalization component and an extension component.

Abstract: Disclosed are compositions and methods for the multiplexed analysis of one or more intracellular targets of a single cell. Exemplary compositions of the disclosure comprise a surface comprising a plurality of capture agents operatively-linked thereto, wherein each capture agent specifically binds to a distinct intracellular target and wherein the plurality of capture agents form a repeating pattern; a substrate comprising a plurality of chambers, wherein the substrate releasably couples with the surface and wherein each chamber of the plurality of chambers comprises at least one repeat of the repeating pattern of the plurality of capture agents of the surface; a coating composition comprising a cell lysis composition; and a linker composition comprising a functionalization component and an extension component.

Abstract: Methods for selectively combining discrete entities including, e.g. cells, reagents, drugs, hydrogels, extracellular matrices beads, particles, biological materials, media, or a combination thereof, are provided. In certain aspects, the methods include sorting a plurality of discrete entities and trapping two or more discrete entities for a time sufficient for the two or more discrete entities to combine to form a combined discrete entity. In certain aspects, the methods include making the plurality of discrete entities. In certain aspects, the methods include detecting or analyzing the discrete entities, e.g. via optical detection. In certain aspects, the methods include manipulating or analyzing the combined discrete entity or a component therein, e.g. imaging, sequencing, culturing, e.g., three-dimensional culturing, and measuring cell-cell interactions. Systems and devices for practicing the subject methods are also provided.

Abstract: The present disclosure provides methods, compositions, and systems employing blocked primers. Aspects of the disclosure include providing a blocked primer reaction mixture that includes a blocked primer and a template nucleic acid component from a single cell; unblocking the blocked primer to produce an active primer reaction mixture and subjecting the activated primer reaction mixture to primer extension conditions, such as nucleic acid amplification conditions.

Abstract: The present invention provides compositions, systems, and methods for barcoding cells, beads, and secreted proteins in discrete entities (e.g. droplets) to allow sequencing data from such components that are separated during processing to be associated via the common barcodes. In some embodiments, the barcodes are tethered to the cell surface via a lipid, cholesterol, or antibody, or are attached to a surface molecule that moves from one cell to another via trogocytosis. In certain embodiments, such methods allow cell-cell interactions or secreted proteins in the discrete entity to be monitored.

Abstract: The present disclosure provides methods, device, assemblies, and systems for dispensing and visualizing single cells. For example, provided herein are systems and methods for dispensing a dispense volume into a plurality of wells of a multi-well device, where, on average, a pre-determined number of cells (e.g., 1-20) are present in the dispense volume, and determining, via a cellular label, the number of cells present in each of the plurality of wells. Such dispensing and cell detection may be repeated a number of times with respect to wells identified as having less than the pre-determined number of cells in order increase the number wells in the multi-well device containing the desired number (e.g., a single cell).

Abstract: The present disclosure provides methods, device, assemblies, and systems for dispensing and visualizing single cells. For example, provided herein are systems and methods for dispensing a dispense volume into a plurality of wells of a multi-well device, where, on average, a pre-determined number of cells (e.g., 1-20) are present in the dispense volume, and determining, via a cellular label, the number of cells present in each of the plurality of wells. Such dispensing and cell detection may be repeated a number of times with respect to wells identified as having less than the pre-determined number of cells in order increase the number wells in the multi-well device containing the desired number (e.g., a single cell).

Abstract: Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention.

Abstract: The present invention provides systems, kits, and methods for analyzing cell-cell interactions, such as transmembrane proteins binding to surface displayed variable regions, via discrete entity (e.g., droplet) microfluidics. In certain embodiments, a plurality of first discrete entities and a plurality of second discrete entities are merged on a substrate to generate a plurality of merged fixed entities (e.g., fixed via an electrical force), each of which contains one cell expressing a transmembrane (TM) protein and labeled clonal cells displaying a heterologous antibody variable region. In certain embodiments, any binding of the clonal cells to the TM expressing cell is detected in each merged fixed entity, and the clonal cells found to bind are treated in order to sequence the nucleic acid encoding the variable region.

Abstract: The present disclosure provides methods, device, assemblies, and systems for dispensing and visualizing single cells. For example, provided herein are systems and methods for dispensing a dispense volume into a plurality of wells of a multi-well device, where, on average, a pre-determined number of cells (e.g., 1-20) are present in the dispense volume, and determining, via a cellular label, the number of cells present in each of the plurality of wells. Such dispensing and cell detection may be repeated a number of times with respect to wells identified as having less than the pre-determined number of cells in order increase the number wells in the multi-well device containing the desired number (e.g., a single cell).

Abstract: Provided herein are compositions and methods for identifying cancer cells. In particular, provided herein are assays for identifying copy number variations (e.g., in circulating tumor cells (CTC)) indicative of cancer (e.g., lymphoma).

Abstract: The present disclosure provides methods, device, assemblies, and systems for dispensing and visualizing single cells. For example, provided herein are systems and methods for dispensing a dispense volume into a plurality of wells of a multi-well device, where, on average, a pre-determined number of cells (e.g., 1-20) are present in the dispense volume, and determining, via a cellular label, the number of cells present in each of the plurality of wells. Such dispensing and cell detection may be repeated a number of times with respect to wells identified as having less than the pre-determined number of cells in order increase the number wells in the multi-well device containing the desired number (e.g., a single cell).

Abstract: Disclosed are compositions and methods for the multiplexed analysis of one or more intracellular targets of a single cell. Exemplary compositions of the disclosure comprise a surface comprising a plurality of capture agents operatively-linked thereto, wherein each capture agent specifically binds to a distinct intracellular target and wherein the plurality of capture agents form a repeating pattern; a substrate comprising a plurality of chambers, wherein the substrate releasably couples with the surface and wherein each chamber of the plurality of chambers comprises at least one repeat of the repeating pattern of the plurality of capture agents of the surface; a coating composition comprising a cell lysis composition; and a linker composition comprising a functionalization component and an extension component.

Abstract: Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention.