Single‐Calibration Cell Size Measurement With Flow Cytometry

Cytometry Part A, Journal Year: 2025, Volume and Issue: unknown

Published: March 12, 2025

ABSTRACT Measuring the size of individual cells in high‐throughput experiments is often important biomedical research and applications. Nevertheless, popular tools for single‐cell biology, such as flow cytometers, only offer proxies a cell's size, typically reported arbitrary scales subject to changes instrument's settings selected by multiple users. In this paper, we demonstrate that it possible calibrate flowcytometry laser scatter signals with accurate measures cell diameter from separate devices calibration can be conserved upon settings. We our approach based on cytometric sorting mammalian line according selection parameters, followed determination Coulter counter. A straightforward procedure presented relates parameters absolute measurements using linear models, along transformation converts between different instrument cytometer. Our method makes record cytometer units correlate other features are recorded parallel fluorescence detection channels.

Language: Английский

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Uncovering Cell Cycle-Dependent Effects on Cell Survival in Near-Infrared Photoimmunotherapy

Experimental Cell Research, Journal Year: 2025, Volume and Issue: 448(2), P. 114570 - 114570

Published: April 22, 2025

Near-infrared photoimmunotherapy (NIR-PIT) is an innovative cancer treatment that selectively induces cell death in cells. Cetuximab-IRdye700DX (Cmab-IR700) conjugate commonly used NIR-PIT for head and neck squamous carcinoma (HNSCC) because of the frequent overexpression epidermal growth factor receptor (EGFR) HNSCC This study examined influence cycle phases on response sensitivity to lines expressing a fluorescent ubiquitination-based indicator (Fucci). The timing was quantified using time-lapse imaging clonogenic assay assess survival. results indicated varied among lines, with G1-phase cells HSC3 CAL33 showing slower than those S/G2/M phases, whereas HeLa exhibited no phase-dependent correlation. Cell rupture predominant cells, combination swelling. Clonogenic survival differed mirroring variations death. Among demonstrated greater resistance NIR-PIT. EGFR expression levels, which according line phase, were associated Additionally, L-ascorbic acid-treated increased time reduced sensitivity, may be due reactive oxygen species. These findings provide information development strategies based kinetics enhance therapeutic outcomes.

Language: Английский

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Colony context and size-dependent compensation mechanisms give rise to variations in nuclear growth trajectories

Cell Systems, Journal Year: 2025, Volume and Issue: unknown, P. 101265 - 101265

Published: May 1, 2025

To investigate how cellular variations arise across spatiotemporal scales in a population of identical healthy cells, we performed data-driven analysis nuclear growth hiPS cell colonies as model system. We generated 3D timelapse dataset thousands nuclei over multiple days and developed open-source tools for image data feature-based exploration. Together, these data, tools, workflows comprise framework systematic quantitative dynamics at individual levels, the further highlights important aspects to consider when interpreting data. found that volume trajectories from short-timescale attributable their context within colony. identified time-invariant compensation relationship between duration starting population. Notably, discovered inheritance plays crucial role determining two key features while other are determined by not inherited.

Language: Английский

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A Generalized Adder mechanism for Cell Size Homeostasis: Implications for Stochastic Dynamics of Clonal Proliferation

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 19, 2024

Abstract Measurements of cell size dynamics have revealed phenomeno-logical principles by which individual cells control their across diverse organisms. One the emerging paradigms homeostasis is adder , where cycle duration established such that increase from birth to division independent newborn size. We provide a mechanistic formulation considering follows any arbitrary non-exponential growth law . Our results show main requirement obtain an regardless (the time derivative size) regulators are produced at rate proportional and triggered when these molecules reach prescribed threshold level. Among implications this generalized adder, we investigate fluctuations in proliferation single-cell derived colonies. Considering exponential growth, random clonal transient then eventually decay zero over (i.e., populations become asymptotically more similar). In contrast, several forms (with adder-based control) yield qualitatively different results: monotonically reaching non-zero value. These characterize interplay between level population level, explaining broad sizes seen barcoded human lines.

Language: Английский

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The cell cycle inhibitor RB is diluted in G1 and contributes to controlling cell size in the mouse liver

Frontiers in Cell and Developmental Biology, Journal Year: 2022, Volume and Issue: 10

Published: Aug. 25, 2022

Every type of cell in an animal maintains a specific size, which likely contributes to its ability perform physiological functions. While some size control mechanisms are beginning be elucidated through studies cultured cells, it is unclear if and how such animal. For example, was recently shown that RB, the retinoblastoma protein, diluted by growth G1 promote size-dependence G1/S transition. However, remains what extent RB-dilution mechanism controls We therefore examined contribution mouse liver. Consistent with model, genetic perturbations decreasing RB protein concentrations inducible shRNA expression or liver-specific Rb1 knockout reduced hepatocyte while increasing Fah −/− model increased size. Moreover, concentration reflects as lower larger hepatocytes. In contrast, cycle activators Cyclin D1 E2f1 were relatively constant. Lastly, loss weakened control, i.e. , inverse correlation between much cells grew large they at birth. Taken together, our results show liver linking

Language: Английский

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Recent Developments in Small-Molecule Fluorescent Probes for Cellular Senescence

Chemosensors, Journal Year: 2024, Volume and Issue: 12(7), P. 141 - 141

Published: July 15, 2024

Cellular senescence is a recently emerged research topic in modern biology. Often described as double-edged sword, it encompasses numerous essential biological processes, including beneficial effects such wound healing and embryonic development, well detrimental contributions to chronic inflammation tumor development. Consequently, there an increasing need unravel the intricate networks of develop reliable detection methods distinguish from related phenomena. To address these challenges, variety have been developed. In particular, small-molecule fluorescent probes offer distinct advantages suitability for real-time live cell monitoring vivo imaging, superior tunable properties, versatile applications. this review, we explored recent advancements development toward cellular by targeting various senescence-related These phenomena include upregulation senescence-associated enzymes, perturbation subcellular environment, increased endogenous ROS levels. Moreover, multi-senescence biomarker-targeting approaches are also discussed improve their sensitivities specificities senescence. With advances probe current challenges field facilitate further progress.

Language: Английский

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Mechanisms of growth-dependent regulation of the Gin4 kinase

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 21, 2024

Cell cycle progression is dependent upon cell growth. Cells must therefore translate growth into a proportional signal that can be used to determine when there has been sufficient for progression. In budding yeast, the protein kinase Gin4 required normal control of and undergoes gradual hyperphosphorylation activation are extent growth, which suggests could function in mechanisms measure However, molecular drive poorly understood. Here, we biochemical reconstitution genetic analysis test hypotheses phosphorylation Gin4. We ruled out previous model phosphatidylserine delivered sites plasma membrane binds initiate autophosphorylation. Instead, show Elm1, homolog mammalian Lkb1 tumor suppressor kinase, promote vitro, likely via initiation Furthermore, casein I growth-dependent also regulation Elm1. Together, these discoveries lead new insight link

Language: Английский

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The G1/S transition is promoted by Rb degradation via the E3 ligase UBR5

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Oct. 4, 2023

Mammalian cells make the decision to divide at G1/S transition in response diverse signals impinging on retinoblastoma protein Rb, a cell cycle inhibitor and tumor suppressor. Rb is inhibited by two parallel pathways. In canonical pathway, Cyclin D-Cdk4/6 kinase complexes phosphorylate inactivate Rb. second, recently discovered Rb's concentration decreases during G1 promote progressing through transition. However, mechanisms underlying this second pathway are unknown. Here, we found that drop recovery S/G2 controlled phosphorylation-dependent degradation. early phase, un- hypo-phosphorylated targeted E3 ligase UBR5.

Language: Английский

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Augmented Doubly Robust Post-Imputation Inference for Proteomic data

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: March 27, 2024

Quantitative measurements produced by mass spectrometry proteomics experiments offer a direct way to explore the role of proteins in molecular mechanisms. However, analysis such data is challenging due large proportion missing values. A common strategy address this issue utilize an imputed dataset, which often introduces systematic bias into down-stream analyses if imputation errors are ignored. In paper, we propose statistical framework inspired doubly robust estimators that offers valid and efficient inference for proteomic data. Our combines powerful machine learning tools, as variational autoencoders, augment quality with high-dimensional peptide data, parametric model estimate propensity score debiasing outcomes. estimator compatible double has provable properties. Simulation studies verify its empirical superiority over other existing procedures. application both single-cell bulk-cell Alzheimer's Disease our method utilizes gain additional, meaningful discoveries yet maintains good control false positives.

Language: Английский

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Single-cell Growth Rate Variability in Balanced Exponential Growth

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: June 25, 2024

Exponential accumulation of cell size and highly expressed proteins is observed in many bacterial species at the single level. rates exhibit cycle-by-cycle fluctuations correlation across components - different size. In such balanced growth, homeostasis all variables maintained simultaneously. this study, we examine phenomenological features growth-rate variability present a theoretical framework to explain them emergence multi-variable homeostasis. Our findings suggest that results from high-dimensional dynamic attractor supporting exponential growth. The stability leads decay instantaneous growth rate noise throughout cycle, aligning with empirical findings. We also correctly predict cells higher experience faster noise. Surprisingly, our analysis identifies generated by uneven division as primary source rates. theory offers clear explanation for observations, validated against extensive single-cell data. spontaneously interactions suggests specific control mechanisms correcting deviations target may be unnecessary.

Language: Английский

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