A flow cytometry based approach to identify distinct coelomocyte subsets of the purple sea urchin, Strongylocentrotus purpuratus

Hudgell, Megan A. Barela; Grayfer, Leon; Smith, L. Courtney

doi:10.1016/j.dci.2022.104352

Cited by 4 publications

(9 citation statements)

References 37 publications

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“…CF (200 μL) was withdrawn from sea urchins according to ( 42 , 43 ). This sampling approach evaluates the coelomocytes that are in the coelomic fluid, which does not include coelomocytes in peripheral tissues.…”

Section: Methodsmentioning

confidence: 99%

“…The diluted CF was held on ice for up to one hour prior to further use. Coelomocyte concentration was estimated for individual sea urchins prior to CF collection and dilution according to previous reports ( 36 , 43 ).…”

Section: Methodsmentioning

confidence: 99%

“…Vibrio diazotrophicus (American Type Culture Collection; item #33466) ( 47 ) was grown in marine broth (Difco Laboratories) as described ( 43 , 48 , 49 ). Vibrio cells were re-suspended in artificial CF (aCF; 10 mM CaCl 2, 14 mM KCL, 50 mM MgCl 2 , 390 mM NaCl, 1.7 mM Na 2 HCO 3, 25 mM Na 2 SO 4 ( 45 ), adjusted to 10 4 and 10 6 bacteria/µL in 1 mL aliquots, heat-killed and stored at -20°C.…”

Section: Methodsmentioning

confidence: 99%

“…Sea urchins were injected initially with 10 4 heat-killed V. diazotrophicus per ml of CF on day 2 and 10 6 V . diazotrophus per ml of CF on day 5 as reported ( 43 ), or with zymosan A, or vehicle (aCF) by injection through the peristomial membrane as described ( 42 ). The number of V. diazotrophicus cells/ml or zymosan A particles/mL that were injected was based on the estimated volume of the CF for each animal according to ( 50 ).…”

Section: Methodsmentioning

confidence: 99%

“…To improve the analysis and quantification of sea urchin coelomocytes and to circumvent problems of cell binding (or not binding) to slides followed by manual cell counts, we reported the identification of different cell populations by differential flow cytometry gating strategies. Gates were established and optimized by evaluating fractions of coelomocyte types separated by density centrifugation to identify the large phagocytes that are a mixture of discoidal and polygonal phagocytes, small phagocytes, RSCs, and a mixture of vibratile cells and CSCs ( 43 ). By using these gates, accurate quantification of changes in coelomocyte populations can be explored in response to various challenges.…”

Section: Introductionmentioning

confidence: 99%

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Coelomocyte populations in the sea urchin, Strongylocentrotus purpuratus, undergo dynamic changes in response to immune challenge

2022

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The sea urchin, Strongylocentrotus purpuratus has seven described populations of distinct coelomocytes in the coelomic fluid that are defined by morphology, size, and for some types, by known functions. Of these subtypes, the large phagocytes are thought to be key to the sea urchin cellular innate immune response. The concentration of total coelomocytes in the coelomic fluid increases in response to pathogen challenge. However, there is no quantitative analysis of how the respective coelomocyte populations change over time in response to immune challenge. Accordingly, coelomocytes collected from immunoquiescent, healthy sea urchins were evaluated by flow cytometry for responses to injury and to challenge with either heat-killed Vibrio diazotrophicus, zymosan A, or artificial coelomic fluid, which served as the vehicle control. Responses to the initial injury of coelomic fluid collection or to injection of V. diazotrophicus show significant increases in the concentration of large phagocytes, small phagocytes, and red spherule cells after one day. Responses to zymosan A show decreases in the concentration of large phagocytes and increases in the concentration of small phagocytes. In contrast, responses to injections of vehicle result in decreased concentration of large phagocytes. When these changes in coelomocytes are evaluated based on proportions rather than concentration, the respective coelomocyte proportions are generally maintained in response to injection with V. diazotrophicus and vehicle. However, this is not observed in response to zymosan A and this lack of correspondence between proportions and concentrations may be an outcome of clearing these large particles by the large phagocytes. Variations in coelomocyte populations are also noted for individual sea urchins evaluated at different times for their responses to immune challenge compared to the vehicle. Together, these results demonstrate that the cell populations in sea urchin immune cell populations undergo dynamic changes in vivo in response to distinct immune stimuli and to injury and that these changes are driven by the responses of the large phagocyte populations.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Coelomocyte populations in the sea urchin, Strongylocentrotus purpuratus, undergo dynamic changes in response to immune challenge

2022

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Interaction between nanomaterials and the innate immune system across evolution

et al. 2023

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Interaction of engineered nanomaterials (ENMs) with the immune system mainly occurs with cells and molecules of innate immunity, which are present in interface tissues of living organisms. Immuno-nanotoxicological studies aim at understanding if and when such interaction is inconsequential or may cause irreparable damage. Since innate immunity is the first line of immune reactivity towards exogenous agents and is highly conserved throughout evolution, this review focuses on the major effector cells of innate immunity, the phagocytes, and their major sensing receptors, Toll-like receptors (TLRs), for assessing the modes of successful versus pathological interaction between ENMs and host defences. By comparing the phagocyte-and TLR-dependent responses to ENMs in plants, molluscs, annelids, crustaceans, echinoderms and mammals, we aim to highlight common recognition and elimination mechanisms and the general sufficiency of innate immunity for maintaining tissue integrity and homeostasis.

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Lipofection mediated transfection fails for sea urchin coelomocytes

Hudgell

Smith

2022

PLoS ONE

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Molecular cloning, gene manipulation, gene expression, protein function, and gene regulation all depend on the introduction of nucleic acids into target cells. Multiple methods have been developed to facilitate such delivery including instrument based microinjection and electroporation, biological methods such as transduction, and chemical methods such as calcium phosphate precipitation, cationic polymers, and lipid based transfection, also known as lipofection. Here we report attempts to lipofect sea urchin coelomocytes using DOTAP lipofection reagent packaged with a range of molecules including fluorochromes, in addition to expression constructs, amplicons, and RNA encoding GFP. DOTAP has low cytotoxicity for coelomocytes, however, lipofection of a variety of molecules fails to produce any signature of success based on results from fluorescence microscopy and flow cytometry. While these results are negative, it is important to report failed attempts so that others conducting similar research do not repeat these approaches. Failure may be the outcome of elevated ionic strength of the coelomocyte culture medium, uptake and degradation of lipoplexes in the endosomal-lysosomal system, failure of the nucleic acids to escape the endosomal vesicles and enter the cytoplasm, and difficulties in lipofecting primary cultures of phagocytic cells. We encourage others to build on this report by using our information to optimize lipofection with a range of other approaches to work towards establishing a successful method of transfecting adult cells from marine invertebrates.

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A flow cytometry based approach to identify distinct coelomocyte subsets of the purple sea urchin, Strongylocentrotus purpuratus

Cited by 4 publications

References 37 publications

Coelomocyte populations in the sea urchin, Strongylocentrotus purpuratus, undergo dynamic changes in response to immune challenge

Coelomocyte populations in the sea urchin, Strongylocentrotus purpuratus, undergo dynamic changes in response to immune challenge

Interaction between nanomaterials and the innate immune system across evolution

Lipofection mediated transfection fails for sea urchin coelomocytes

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