Optimizing Optical Flow Cytometry for Cell Volume-Based Sorting and Analysis

Tzur, Amit; Moore, Jason Z.; Jorgensen, Paul; Shapiro, Howard M.; Kirschner, Marc W.

doi:10.1371/journal.pone.0016053

Cited by 171 publications

(165 citation statements)

References 18 publications

Supporting

Mentioning

153

Contrasting

Order By: Relevance

“…Further, this phenotype was accentuated in LR-MDS when compared with normal donors (P 5 6.0 3 10 25 ), whereas there were no significant differences in HR-MDS. These findings were corroborated using side scatter area (SSC-A) flow cytometry measurements, a validated reference for cell size, 29 in ungated BM-MNCs from LR-MDS patients (n 5 7) and controls (n 5 6), as well as in antigenically distinct hematopoietic lineages ( Figure 3B). 29 Finally, mean NLRP3 MFI correlated with mean cell area in LR-MDS patients, but not HR-MDS patients (r 5 0.49) (P 5 7.8 3 10 23 ) ( Figure 3C).…”

Section: The Alarmin S100a9 Initiates Pyroptosissupporting

confidence: 53%

“…These findings were corroborated using side scatter area (SSC-A) flow cytometry measurements, a validated reference for cell size, 29 in ungated BM-MNCs from LR-MDS patients (n 5 7) and controls (n 5 6), as well as in antigenically distinct hematopoietic lineages ( Figure 3B). 29 Finally, mean NLRP3 MFI correlated with mean cell area in LR-MDS patients, but not HR-MDS patients (r 5 0.49) (P 5 7.8 3 10 23 ) ( Figure 3C). To assess pore formation, influx of the membrane-impermeable cationic dye ethidium bromide (EtBr) was assessed by flow cytometry.…”

Section: The Alarmin S100a9 Initiates Pyroptosissupporting

confidence: 53%

See 1 more Smart Citation

The NLRP3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype

Basiorka¹,

McGraw²,

Eksioglu³

et al. 2016

Blood

275

350

View full text Add to dashboard Cite

Key Points• Key biological features of MDSs are explained by NLRP3 inflammasome activation, which drives pyroptotic cell death and b-catenin activation.• Alarmin signals and founder gene mutations license this redox-sensitive inflammasome platform.Despite genetic heterogeneity, myelodysplastic syndromes (MDSs) share features of cytological dysplasia and ineffective hematopoiesis. We report that a hallmark of MDSs is activation of the NLRP3 inflammasome, which drives clonal expansion and pyroptotic cell death. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPCs) overexpress inflammasome proteins and manifest activated NLRP3 complexes that direct activation of caspase-1, generation of interleukin-1b (IL-1b) and IL-18, and pyroptotic cell death. Mechanistically, pyroptosis is triggered by the alarmin S100A9 that is found in excess in MDS HSPCs and bone marrow plasma. Further, like somatic gene mutations, S100A9-induced signaling activates NADPH oxidase (NOX), increasing levels of reactive oxygen species (ROS) that initiate cation influx, cell swelling, and b-catenin activation. Notably, knockdown of NLRP3 or caspase-1, neutralization of S100A9, and pharmacologic inhibition of NLRP3 or NOX suppress pyroptosis, ROS generation, and nuclear b-catenin in MDSs and are sufficient to restore effective hematopoiesis. Thus, alarmins and founder gene mutations in MDSs license a common redox-sensitive inflammasome circuit, which suggests new avenues for therapeutic intervention. (Blood. 2016;128(25):2960-2975

show abstract

Section: The Alarmin S100a9 Initiates Pyroptosissupporting

confidence: 53%

Section: The Alarmin S100a9 Initiates Pyroptosissupporting

confidence: 53%

The NLRP3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype

Basiorka¹,

McGraw²,

Eksioglu³

et al. 2016

Blood

275

350

View full text Add to dashboard Cite

show abstract

“…Throughout their experiments, the authors used flow cytometry to measure cell size using forward scatter (FSC). Calibration of FSC is important for its rigorous interpretation [32]. Upon binning cells by diameter, with bin width of $100 nm, the authors found good correlation between FSC and cell radius across the binned populations.…”

Section: Linking Mitochondrial Heterogeneity and Cell Sizementioning

confidence: 99%

Mitochondrial heterogeneity, metabolic scaling and cell death

et al. 2017

View full text Add to dashboard Cite

Heterogeneity in mitochondrial content has been previously suggested as a major contributor to cellular noise, with multiple studies indicating its direct involvement in biomedically important cellular phenomena. A recently published dataset explored the connection between mitochondrial functionality and cell physiology, where a non-linearity between mitochondrial functionality and cell size was found. Using mathematical models, we suggest that a combination of metabolic scaling and a simple model of cell death may account for these observations. However, our findings also suggest the existence of alternative competing hypotheses, such as a non-linearity between cell death and cell size. While we find that the proposed non-linear coupling between mitochondrial functionality and cell size provides a compelling alternative to previous attempts to link mitochondrial heterogeneity and cell physiology, we emphasise the need to account for alternative causal variables, including cell cycle, size, mitochondrial density and death, in future studies of mitochondrial physiology.

show abstract

“…Tight gating of both forward and side scatter was performed to restrict the size heterogeneity of the population as well as eliminate cell debris and cell aggregates. Gating removed greater than 60% of the cell population, resulting in forward scatter pulse width variance (seen to correlate well with cell volume [4]), with a coefficient of variation (CV) of 0.06. The same gating settings were used to isolate cells from both infections.…”

Section: Cells and Virusesmentioning

confidence: 99%

Single-Cell Analysis Uncovers Extensive Biological Noise in Poliovirus Replication

Schulte

Andino

2014

J Virol

View full text Add to dashboard Cite

Viral infections often begin with a very small number of initiating particles. Accordingly, the outcome of an infection is likely to be affected by variability in the initial molecular interactions between virus and host. In this study, we investigated the range of outcomes upon infection of single cells. We isolated individual cells infected with poliovirus at low or high multiplicities of infection (MOI) and measured viral genomic replication and infectious viral progeny in each cell. We first determined that at 7 h postinfection, the ratio of positive to negative strands in individual cells varies from 5:1 to more than 190:1, with and average of 20:1, suggesting a significant variability in RNA synthesis. We further found that while virus genome production is higher in cells infected at a high multiplicity, the production of infectious particles is largely independent of the number of viruses infecting each cell. Strikingly, by correlating RNA and particle production within individual infections, we uncovered a significant contribution of stochastic noise to the outcome of infection. At low MOI, stochastic influences appear as kinetic effects which are most critical at the initial steps in infection. At high MOI, stochastic influences appear to dictate the virus's ability to harness cellular resources. We conclude that biological noise is a critical determinant of the overall productivity of viral infections. The distinct nature of stochasticity in the outcome of infection by low and high numbers of viral particles may have important implications for our understanding of the determinants of successful viral infections. IMPORTANCEBy correlating genome and particle production in single-cell infections, we elucidated sources of noise in viral infections. When a cell was infected by only a single infectious particle, variation in the kinetics of the initial steps of replication contributed significantly to the overall productivity of the infection. Additionally, variation in the distribution of subcellular resources impacted infections initiated by one or many infectious particles. We also observed that when a cell was infected with multiple particles, more genomes were produced, while particle production was hindered by an apparent cellular resource limit. Understanding variations in viral infections may illuminate the dynamics of infection and pathogenesis and has implications for virus adaptation and evolution.

show abstract

Optimizing Optical Flow Cytometry for Cell Volume-Based Sorting and Analysis

Cited by 171 publications

References 18 publications

The NLRP3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype

The NLRP3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype

Mitochondrial heterogeneity, metabolic scaling and cell death

Single-Cell Analysis Uncovers Extensive Biological Noise in Poliovirus Replication

Contact Info

Product

Resources

About