Validated flow cytometry allows rapid quantitative assessment of immune responses in amphibians

in Amphibia-Reptilia
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Assessments of amphibian immune state have been commonly made through indirect methods like phytohemagglutinin (PHA) injections, or by direct methods like cell counts from blood smears. Here we validate a simple method to assess immune responses in amphibians by means of flow cytometry with a fluorescent lipophilic dye (3,3′ Dipentyloxacarbocyanine), which removes the need for specific antibodies. We experimentally altered the immunological state of Pelobates cultripes tadpoles by exposing some to exogenous corticosterone. We then determined the immune state of each tadpole through both blood smears and flow cytometry. We found that both techniques showed similar patterns of the proportion of white blood cells. Once validated, flow cytometry also allowed quantitation of changes in absolute number of white cells. We discuss the suitability of both techniques attending to the accuracy of each technique, body size requirements, or the tractability in field studies.

Validated flow cytometry allows rapid quantitative assessment of immune responses in amphibians

in Amphibia-Reptilia

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Figures

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    (A) Gates of Pelobates cultripes blood cell populations revealed by their side scatter (SSC-HLin) and green fluorescence (GRN-HLin) properties. Erythrocytes, granulocytes, lymphocytes, and monocytes were designed as E, G, L, and M, respectively. In this example 24 073 erythrocytes, 202 lymphocytes, 146 granulocytes, and 4 monocytes were quantified. (B) Example of a lymphocyte gate. Gates were optimized applying a gating strategy based on forward scatter (FSC-HLin) and green fluorescence (GRN-HLin) dot plot combination.

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    (A) Granulocytes:Lymphocytes (G:L) ratio determined by flow cytometry in response to no exogenous (0 nM CORT) and to 100 nM corticosterone (100 nM CORT, stress induction) treatments. Bars indicate ±S.E. (B) G:L ratio determined by blood smears in response to 0 and 100 nM corticosterone treatments. (C) Correlation between the G:L ratio determined by flow cytometry or by direct cell count in blood smears (correlation coefficient = 0.61). (D) Absolute count of lymphocytes (white) and granulocytes (grey) in response to 0 and 100 nM corticosterone treatments as determined by flow cytometry.

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