Apoptotic cell mimic
Inventors
Ahn, Keunho • DENG, Zhenyu • DE GEUS, Martina • Nguyen, Anh Tuan
Assignees
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Publication Number
US-12332241-B2
Publication Date
2025-06-17
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 comprising multiple engineered hydrogel bead populations for use with flow cytometry live/dead and apoptosis assays. The kit includes a first population of hydrogel beads, a second population of hydrogel beads, and a third population of hydrogel beads. The engineered beads comprise polymerized monomer and carry annexin V and double stranded DNA combinations corresponding to apoptosis-related signals and DNA staining signals.
In the first population, hydrogel beads comprise annexin V and double stranded DNA together with a polymerized monomer. In the second population, hydrogel beads comprise annexin V but lack the double stranded DNA of the first population. In the third population, hydrogel beads lack both annexin V and double stranded DNA. The arrangement creates multiple bead populations that represent different combinations of pre-apoptotic/apoptotic binding and nucleic-acid staining.
The annexin V of the first and/or second population comprises SEQ ID NO: 102. The disclosed bead-population design supports distinguishing signal combinations associated with apoptosis-related markers and DNA dyes, while providing viability controls and discrimination for live, apoptotic, and dead populations. The disclosed approach is directed to improving gating, compensation, and spectral-unmixing performance for flow cytometry compared with cells or polystyrene beads.
Claims Coverage
The independent claim defines a kit with three hydrogel bead populations that collectively cover three signal-combination states: annexin V plus double stranded DNA, annexin V without double stranded DNA, and polymerized monomer without annexin V and without double stranded DNA. The claim requires annexin V of the first and/or second population to comprise SEQ ID NO: 102.
Three hydrogel bead populations with annexin V and double stranded DNA combinations
A kit comprising a first population of hydrogel beads with polymerized monomer, annexin V, and double stranded DNA; a second population with polymerized monomer and annexin V but lacking the double stranded DNA; and a third population with polymerized monomer lacking annexin V and lacking double stranded DNA.
SEQ ID NO: 102 annexin V in the first and/or second population
The annexin V of the first and/or second population of hydrogel beads comprises SEQ ID NO: 102.
The independent claim centers on a multi-population hydrogel bead kit that separates annexin V-related signal presence from double stranded DNA-associated signal presence, while requiring SEQ ID NO: 102 for the annexin V present in the first and/or second population.
Stated Advantages
Improved discrimination for live/apoptotic/dead using first/second/third hydrogel bead populations.
Reduced non-specific binding compared with polystyrene beads and improved viability control performance relative to heat-killed cells and polystyrene beads.
Improved gating, compensation, and spectral unmixing versus single-dye-stained live cells and cell-based controls.
Bead stability for at least 37 days at 4°C.
Configurable optical-scatter similarity to biological cell types including granulocytes, monocytes, and lymphocytes.
Reduced variability/time versus heat/chemical induction of apoptosis [procedural detail omitted for safety].
Documented Applications
Flow cytometry live/dead and apoptosis assays using engineered hydrogel bead populations as viability controls and discriminators.
Use as viability control and gating, compensation, and spectral unmixing support in flow cytometry instruments including BD FACSLyric and CYTEK Aurora.
Matching hydrogel beads to forward scatter and side scatter profiles of cell types including granulocytes, monocytes, and lymphocytes.
Flow cytometry control using the hydrogel bead populations for cytometric assays, including examples comparing live/dead/apoptotic bead populations in cytometric assays.
Use of bead populations for improved gating, compensation, and spectral-unmixing in flow cytometry instrumentation.
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