Apoptotic cell mimic
Inventors
Ahn, Keunho • DENG, Zhenyu • DE GEUS, Martina • Nguyen, Anh Tuan
Assignees
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Publication Number
US-12130285-B2
Publication Date
2024-10-29
Expiration Date
Abstract
Provided herein are hydrogel beads that mimic live, dead, and apoptotic cells. The present disclosure also provides kits and compositions of hydrogel beads. The present disclosure further comprises methods of using the kits, compositions, and hydrogel beads to determine if a target cell sample includes one or more live, dead, or apoptotic cells.
Core Innovation
The invention provides a kit of functional hydrogel bead populations for live/dead and apoptosis flow cytometry. The bead populations differentiate by whether they include annexin V and whether they contain an encapsulated nucleic acid that binds an intercalating dye.
A first bead population comprises annexin V and an encapsulated nucleic acid, enabling annexin V-associated pre-apoptotic signal binding and nucleic-acid-associated intercalating dye binding. A second bead population comprises annexin V but lacks the encapsulated nucleic acid, while a third bead population lacks annexin V and lacks the encapsulated nucleic acid, thereby mimicking a signal-negative control population.
The encapsulated nucleic acid of the first population binds an intercalating dye selected from 7-aminoactinomycin D (7AAD) and propidium iodide. The hydrogel beads provide viability-control performance with clearer positive and negative populations and support gating, compensation, and spectral-unmixing controls.
Claims Coverage
The consolidated content includes one independent claim directed to a kit comprising three distinct populations of hydrogel beads that differ by annexin V inclusion and encapsulated nucleic acid inclusion. The independent claim includes three inventive features.
Three differentiated hydrogel bead populations
A kit comprising a first population of hydrogel beads with a polymerized monomer, annexin V, and an encapsulated nucleic acid; a second population with a polymerized monomer and annexin V but lacking the encapsulated nucleic acid; and a third population with a polymerized monomer but lacking annexin V and lacking the encapsulated nucleic acid.
Annexin V sequence specification
Wherein the annexin V of the first and/or second population comprises SEQ ID NO: 102.
Encapsulated nucleic acid intercalating dye binding
Wherein the encapsulated nucleic acid of the first population binds to an intercalating dye selected from 7-aminoactinomycin D (7AAD) and propidium iodide (PI).
The claim coverage centers on a three-population hydrogel bead kit distinguished by annexin V and encapsulated nucleic acid presence or absence, with annexin V specified as SEQ ID NO: 102 and the encapsulated nucleic acid configured to bind 7AAD or propidium iodide.
Stated Advantages
Provides hydrogel viability controls with clearer positive and negative populations.
Improves compensation and spectral unmixing versus cells or polystyrene beads.
Improved viability-control performance versus heat-killed cells and polystyrene beads.
Superior gating/compensation/spectral-unmixing controls compared with cells stained with single dyes.
Allows tuning of optical scatter for matching to target cell types.
Provides bead stability of at least 37 days at 4°C.
Reduced variability and time requirements.
Documented Applications
Flow-cytometry viability controls using beads that mimic live, dead, and pre-apoptotic cells for gating and calibration.
Optical scatter matching for flow-cytometry analysis targeting cell types including lymphocytes, monocytes, and granulocytes.
Live/dead and apoptosis flow cytometry, including viability control and gating/compensation/spectral-unmixing controls.
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