Isolation and Culture of Amphibian (Xenopus laevis) Sub-Capsular Liver and Bone Marrow Cells

Yaparla, Amulya; Grayfer, Leon

doi:10.1007/978-1-4939-8784-9_20

Cited by 4 publications

(6 citation statements)

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“…Bone marrow isolation, culture conditions, and establishment of neutrophil cultures have been previously described 58 . Mast cell cultures were generated according to protocols adapted from Koubourli et al (2018) and Meurer et al (2016) 59, 60 .…”

Section: Methodsmentioning

confidence: 99%

“…Bone marrow cells were isolated as previously described 58 . Briefly, adult X. laevis (approximately one year old) were euthanized in 5% tricaine mesylate followed by cervical dislocation.…”

Section: Supplementary Materialsmentioning

confidence: 99%

“…The X. laevis rSCF, rCSF3 and rIL4 were generated as previously described for rCSF3 57 and as detailed in the supplemental materials. Bone marrow isolation, culture conditions, and establishment of neutrophil cultures have been previously described 58 . Mast cell cultures were generated according to protocols adapted from Koubourli et 59,60 .…”

Section: Recombinant Cytokines and Bone Marrow Granulocyte Culturesmentioning

confidence: 99%

“…The X. laevis rSCF, rCSF3 and rIL4 were generated as previously described for rCSF3 67 and as detailed in the supplemental materials. Bone marrow isolation, culture conditions, and establishment of neutrophil cultures have been previously described 68 . Mast cell cultures were generated according to protocols adapted Cell cultures were maintained at 27°C with 5% CO2 in amphibian serum-free medium supplemented with 10% fetal bovine serum, 0.25% X. laevis serum, 10 μg/mL gentamicin (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, Thermo Fisher Scientific).…”

Section: Recombinant Cytokines and Bone Marrow Granulocyte Culturesmentioning

confidence: 99%

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Amphibian mast cells: barriers to chytrid fungus infections

Hauser,

Garvey,

Crow

et al. 2023

Preprint

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Global amphibian declines are largely driven by deadly disease outbreaks caused by the chytrid fungus,Batrachochytrium dendrobatidis(Bd). In the time since these disease outbreaks were first discovered, much has been learned about the roles of amphibian skin-produced antimicrobial components and skin microbiomes in controllingBd. Yet almost nothing is known about the roles of skin-resident immune cells in anti-Bddefenses. Notably, mammalian mast cells reside within and serve as key immune sentinels in barrier tissues like the skin. Thus, they are critical to immune recognition of pathogens and to orchestrating the ensuing immune responses. Accordingly, we investigated the roles ofXenopus laevisfrog mast cells duringBdinfections. Our findings indicate that enrichment ofX. laevisskin mast cells confers significant anti-Bdprotection and ameliorates the inflammation-associated skin damage caused byBdinfection. Moreover, enrichingX. laevismast cells promotes greater mucin content within cutaneous mucus glands and protects frogs fromBd-mediated changes to their skin microbiomes. Together, this work underlines the importance of amphibian skin-resident immune cells in anti-Bddefenses and introduces a novel approach for investigating amphibian host-chytrid pathogen interactions.

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Section: Methodsmentioning

confidence: 99%

Section: Supplementary Materialsmentioning

confidence: 99%

Section: Recombinant Cytokines and Bone Marrow Granulocyte Culturesmentioning

confidence: 99%

Section: Recombinant Cytokines and Bone Marrow Granulocyte Culturesmentioning

confidence: 99%

See 2 more Smart Citations

Amphibian mast cells: barriers to chytrid fungus infections

Hauser,

Garvey,

Crow

et al. 2023

Preprint

Self Cite

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“…The liver periphery (LP) cells and bone marrow (BM) cells were isolated by a previously described method (30). Briefly, X. laevis femurs and liver tissues were aseptically excised from five individual frogs (N = 5) that had been euthanized by tricaine mesylate overdose and cervical dislocation.…”

Section: Bone Marrow Peripheral Liver and Peripheral Blood Leukocytmentioning

confidence: 99%

Myelopoiesis of the Amphibian Xenopus laevis Is Segregated to the Bone Marrow, Away From Their Hematopoietic Peripheral Liver

Yaparla

Reeves²,

Grayfer

2020

Front. Immunol.

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Across vertebrates, hematopoiesis takes place within designated tissues, wherein committed myeloid progenitors further differentiate toward cells with megakaryocyte/erythroid potential (MEP) or those with granulocyte/macrophage potential (GMP). While the liver periphery (LP) of the Xenopus laevis amphibian functions as a principal site of hematopoiesis and contains MEPs, cells with GMP potential are instead segregated to the bone marrow (BM) of this animal. Presently, using gene expression and western blot analyses of blood cell lineage-specific transcription factors, we confirmed that while the X. laevis LP hosts hematopoietic stem cells and MEPs, their BM contains GMPs. In support of our hypothesis that cells bearing GMP potential originate from the frog LP and migrate through blood circulation to the BM in response to chemical cues; we demonstrated that medium conditioned by the X. laevis BM chemoattracts LP and peripheral blood cells. Compared to LP and by examining a comprehensive panel of chemokine genes, we showed that the X. laevis BM possessed greater expression of a single chemokine, CXCL12, the recombinant form of which was chemotactic to LP and peripheral blood cells and appeared to be a major chemotactic component within BM-conditioned medium. In confirmation of the hepatic origin of the cells that give rise to these frogs' GMPs, we also demonstrated that the X. laevis BM supported the growth of their LP-derived cells.

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Amphibian mast cells serve as barriers to chytrid fungus infections

Hauser,

Garvey,

Crow

et al. 2024

eLife

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Global amphibian declines are largely driven by deadly disease outbreaks caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd). In the time since these disease outbreaks were first discovered, much has been learned about the roles of amphibian skin-produced antimicrobial components and skin microbiomes in controlling Bd. Yet almost nothing is known about the roles of skin-resident immune cells in anti-Bd defenses. Notably, mammalian mast cells reside within and serve as key immune sentinels in barrier tissues like the skin. Thus, they are critical to immune recognition of pathogens and to orchestrating the ensuing immune responses. Accordingly, we investigated the roles of Xenopus laevis frog mast cells during Bd infections. Our findings indicate that enrichment of X. laevis skin mast cells confers significant anti-Bd protection and ameliorates the inflammation-associated skin damage caused by Bd infection. Moreover, enriching X. laevis mast cells promotes greater mucin content within cutaneous mucus glands and protects frogs from Bd-mediated changes to their skin microbiomes. Together, this work underlines the importance of amphibian skin-resident immune cells in anti-Bd defenses and introduces a novel approach for investigating amphibian host-chytrid pathogen interactions.

show abstract

Isolation and Culture of Amphibian (Xenopus laevis) Sub-Capsular Liver and Bone Marrow Cells

Cited by 4 publications

References 9 publications

Amphibian mast cells: barriers to chytrid fungus infections

Amphibian mast cells: barriers to chytrid fungus infections

Myelopoiesis of the Amphibian Xenopus laevis Is Segregated to the Bone Marrow, Away From Their Hematopoietic Peripheral Liver

Amphibian mast cells serve as barriers to chytrid fungus infections

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