![Figure 1. Flow cytometric and gene expression analysis of IgM1:eGFP zebrafish. (A) Map of the zebrafish heavy chain locus with variable (V), diversity (D), joining (J), and constant region exons for IgZ, IgM, and IgD. (B) Organs were isolated from individual fish age 3 to 10 months and analyzed for GFP expression by flow cytometry. The forward scatter and side scatter (FSC/SSC) profile obtained from kidney is shown, with %GFP fluorescence obtained in the lymphocyte (bottom left), precursor (bottom right), and granulocyte (top right) gates. These results are representative of 16 to 22 individual fish from 10 independent experiments. (C) %GFP+ cells in the lymphocyte gate (average plus standard deviation [SD]) of various organs from 2 to 22 individual fish from 2 independent founders of the IgM1:eGFP line (founders 12 and 22) or from lck:GFP reporter fish for comparison. (D) Confocal imaging of GFP+ cells in the skin of the anterior portion of the trunk. (E) Merged image of 4,6 diamidino-2-phenylindole (DAPI) (blue) and anti-GFP (green) staining in the kidney. The image was taken with the ×25 objective of a Leica SP5 inverted confocal microscope. (F) GFP+ cells were sorted to at least 95% purity from the lymphocyte fraction of the kidney, stained with May-Grunwald/Giemsa stain, and photographed at ×1000 (left). More than 95% of the cells exhibited a lymphocytic morphology. Cells sorted from the granulocyte fraction of the kidney are shown for comparison (right, photographed at ×40). These results are a summary of 4 independent experiments. (G) GFP− and GFP+ cells were sorted from the lymphocyte (KL–, KL+) or precursor (KP−, KP+) fraction of the kidney or the lymphocyte fraction of the spleen (SL−, SL+) from 2 to 3 fish and analyzed by qRT-PCR. Average expression plus standard error of the mean (SEM) of IgM, IgZ, or lck relative to their expression in adult kidney (ΔΔCt) is shown. These results are a summary of 3 independent experiments. (H) GFP+ cells were sorted from the lymphocyte (KL+) or precursor (KP+) fraction of the kidneys from 2 to 3 fish. Average expression plus SD of IgM, mpx, gata1, and cd41 relative to their expression in adult kidney is shown. These results are a summary of 2 independent experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/122/8/10.1182_blood-2012-12-471029/4/e1f1.jpeg?Expires=1724240042&Signature=NDmA6OjSSvI5qAwAIUqKSRHy2eUUvmAJTkJo807zE6Mi~oU7E4AMB-f5QhE9-gBF2znBvSbUyOfGES25mtG8Umfeu2WGdj4e-9Yqj5Qx~aVCSVaHuyTZdrswgEo8DaT466km4jXSsVSmXXuLIhQWHZm70xPvu~v3w5wZjbIQFUObEvgZZZmErAzVXmw0aZD5NSU-ztYDJ4ey0sq7kDcVRp~PvWwU7UnY6kLpMUHRi-H0IiptrSeUNrMhg0XWldTde9wPyYlD~0JEsZ~gpNvEEEHhN0XG5-eAv0HBz2-OTVlDmoAReOeJ8t5kX1CuyfLXKiXV5xA7Lvl2h6Kg0yiOLg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Flow cytometric and gene expression analysis of IgM1:eGFP zebrafish. (A) Map of the zebrafish heavy chain locus with variable (V), diversity (D), joining (J), and constant region exons for IgZ, IgM, and IgD. (B) Organs were isolated from individual fish age 3 to 10 months and analyzed for GFP expression by flow cytometry. The forward scatter and side scatter (FSC/SSC) profile obtained from kidney is shown, with %GFP fluorescence obtained in the lymphocyte (bottom left), precursor (bottom right), and granulocyte (top right) gates. These results are representative of 16 to 22 individual fish from 10 independent experiments. (C) %GFP+ cells in the lymphocyte gate (average plus standard deviation [SD]) of various organs from 2 to 22 individual fish from 2 independent founders of the IgM1:eGFP line (founders 12 and 22) or from lck:GFP reporter fish for comparison. (D) Confocal imaging of GFP+ cells in the skin of the anterior portion of the trunk. (E) Merged image of 4,6 diamidino-2-phenylindole (DAPI) (blue) and anti-GFP (green) staining in the kidney. The image was taken with the ×25 objective of a Leica SP5 inverted confocal microscope. (F) GFP+ cells were sorted to at least 95% purity from the lymphocyte fraction of the kidney, stained with May-Grunwald/Giemsa stain, and photographed at ×1000 (left). More than 95% of the cells exhibited a lymphocytic morphology. Cells sorted from the granulocyte fraction of the kidney are shown for comparison (right, photographed at ×40). These results are a summary of 4 independent experiments. (G) GFP− and GFP+ cells were sorted from the lymphocyte (KL–, KL+) or precursor (KP−, KP+) fraction of the kidney or the lymphocyte fraction of the spleen (SL−, SL+) from 2 to 3 fish and analyzed by qRT-PCR. Average expression plus standard error of the mean (SEM) of IgM, IgZ, or lck relative to their expression in adult kidney (ΔΔCt) is shown. These results are a summary of 3 independent experiments. (H) GFP+ cells were sorted from the lymphocyte (KL+) or precursor (KP+) fraction of the kidneys from 2 to 3 fish. Average expression plus SD of IgM, mpx, gata1, and cd41 relative to their expression in adult kidney is shown. These results are a summary of 2 independent experiments.
