Functional assessment of automatically sorted pancreatic islets using large particle flow cytometry

Steffen, Anȷa; Ludwig, Barbara; Krautz, Christian; Bornstein, Stefan R.; Solimena, Michele

doi:10.4161/isl.3.5.15939

Cited by 10 publications

(11 citation statements)

References 20 publications

(16 reference statements)

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“…Finally, once islets with low or high glucose sensitivity are identified, they must be rapidly sorted for down-stream analyses or transplantation. While high-throughput islet sorting is currently possible [37] , this technology would have to be integrated with the XF96 spheroid plate in order to maximize applicability of single islet respirometry for islet transplantation.…”

Section: Discussionmentioning

confidence: 99%

Individual islet respirometry reveals functional diversity within the islet population of mice and human donors

Taddeo

Stiles

Sereda

et al. 2018

Molecular Metabolism

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ObjectiveIslets from the same pancreas show remarkable variability in glucose sensitivity. While mitochondrial respiration is essential for glucose-stimulated insulin secretion, little is known regarding heterogeneity in mitochondrial function at the individual islet level. This is due in part to a lack of high-throughput and non-invasive methods for detecting single islet function.MethodsWe have developed a novel non-invasive, high-throughput methodology capable of assessing mitochondrial respiration in large-sized individual islets using the XF96 analyzer (Agilent Technologies).ResultsBy increasing measurement sensitivity, we have reduced the minimal size of mouse and human islets needed to assess mitochondrial respiration to single large islets of >35,000 μm2 area (∼210 μm diameter). In addition, we have measured heterogeneous glucose-stimulated mitochondrial respiration among individual human and mouse islets from the same pancreas, allowing population analyses of islet mitochondrial function for the first time.ConclusionsWe have developed a novel methodology capable of analyzing mitochondrial function in large-sized individual islets. By highlighting islet functional heterogeneity, we hope this methodology can significantly advance islet research.

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Section: Discussionmentioning

confidence: 99%

Individual islet respirometry reveals functional diversity within the islet population of mice and human donors

Taddeo

Stiles

Sereda

et al. 2018

Molecular Metabolism

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“…Furthermore, no functional superiority of small islets was found when in vitro insulin secretion was expressed per islet DNA (Steffen et al. ).…”

Section: Discussionmentioning

confidence: 99%

Influence of organ donor attributes and preparation characteristics on the dynamics of insulin secretion in isolated human islets

Henquin¹

2018

Physiol Rep

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In vitro studies of human pancreatic islets are critical for understanding normal insulin secretion and its perturbations in diabetic β‐cells, but the influence of islet preparation characteristics and organ donor attributes in such experiments is poorly documented. Preparations from normal donors were tested with a standardized protocol evaluating dynamic insulin secretion induced by glucose, tolbutamide, and cAMP (forskolin). Secretion rates, normalized to insulin content (fractional insulin secretion), were analyzed as a function of preparation and donor characteristics. Low purity (25–45%) of the preparation (n = 8) blunted the first phase of insulin secretion induced by glucose or tolbutamide and increased basal secretion, resulting in threefold lower stimulation index than in more pure (55–95%) preparations (n = 43). In these more pure preparations, cold ischemia time (1–13 h) before pancreas digestion did not impact insulin secretion. Islet size (estimated by the islet size index) did not influence the dynamics of secretion, but fractional insulin secretion rates were greater in large than small islets, and positively correlated with islet size. Age of the donors (20–68 years) had no influence on islet size and insulin content or on dynamics and amplitude of insulin secretion, which were also similar in islets from male and female donors. In contrast, islet size and islet insulin content (normalized for size), and basal or stimulated insulin secretion positively correlated with Body‐Mass Index (19–33). These results contradict previous reports on the impact of donor age and islet size and point to possible confounding effects of donor BMI in insulin secretion studies with isolated human islets.

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“…Particles of this size are not able to pass through the injection port and flow cell of most cytometers so specifically designed analysers and sorters are available to identify and separate these organisms as well as, for example, Drosophila melanogaster and Zebrafish embryos . These large particle sorters can also be used for analysing and sorting groups of cells such as imaginal discs, pancreatic islets or embryoid bodies which can be up to 200 μm . Unlike traditional droplet sorters, these sorters use a puff of air to divert particles of interest into a collection receptacle.…”

Section: Cytometric Parametersmentioning

confidence: 99%

Guidelines for the use of flow cytometry and cell sorting in immunological studies^*

et al. 2017

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International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis

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Functional assessment of automatically sorted pancreatic islets using large particle flow cytometry

Cited by 10 publications

References 20 publications

Individual islet respirometry reveals functional diversity within the islet population of mice and human donors

Individual islet respirometry reveals functional diversity within the islet population of mice and human donors

Influence of organ donor attributes and preparation characteristics on the dynamics of insulin secretion in isolated human islets

Guidelines for the use of flow cytometry and cell sorting in immunological studies^*

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Functional assessment of automatically sorted pancreatic islets using large particle flow cytometry

Cited by 10 publications

References 20 publications

Individual islet respirometry reveals functional diversity within the islet population of mice and human donors

Individual islet respirometry reveals functional diversity within the islet population of mice and human donors

Influence of organ donor attributes and preparation characteristics on the dynamics of insulin secretion in isolated human islets

Guidelines for the use of flow cytometry and cell sorting in immunological studies*

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Guidelines for the use of flow cytometry and cell sorting in immunological studies^*