Journal: Frontiers in Veterinary Science
Article Title: Flow cytometric-based detection of CD80 is a useful diagnostic marker of acute myeloid leukemia in dogs
doi: 10.3389/fvets.2024.1405297
Figure Lengend Snippet: Labeling of monocytes, B cells, T cells, and neutrophils isolated from the blood of healthy dogs (representative results from 1 of 2–3 dogs for each cell type) with the anti-CD80 antibody. (A-C) Monocytes, B cells, and T cells were isolated from peripheral blood mononuclear cells (PBMCs) using immunomagnetic beads and anti-CD14-PE, -CD21-FITC, and -CD5-FITC antibodies, respectively. The first panel shows the forward and side scatter events in PBMCs while the second panel shows the forward and side scatter events of isolated cells. The third panel is a fluorescent quadrant plot of double-labeled cells after adding the anti-CD80-APC antibody. The fourth panel shows a representative modified Wright’s-stained image of a cytospin smear of the isolated cells on which 100-cell differential cell counts were done (scale bar = 20 μm). (A) CD14-PE-isolated cells were mostly CD80 + monocytes (84% of a differential cell count), with a few contaminating neutrophils (10%) that were weakly CD14 + (arrows, third and fourth panels). A few monocytes had cytoplasmic vacuoles (fourth panel). Lymphocytes were negative for CD80 − /CD14 − (lower left quadrant, third panel, 6%). (B) CD21-FITC-isolated cells were mostly lymphocytes, which were CD80 − (third panel). Lymphocytes were primarily small cells, some of which had clefted or convoluted nuclei (variants of normal), with a few small or large reactive forms (fourth panel). (C) CD5-FITC-isolated cells were mostly lymphocytes, which were CD80 − (third panel). Lymphocytes were small cells with a few large or reactive forms. Several lymphocytes had a few clear cytoplasmic vacuoles, which could be due to the isolation procedure (fourth panel). (D) Neutrophils were isolated from the 1.077/1.119 interface of the double-density gradient used to obtain PBMCs and were single-labeled with the anti-CD80-APC antibody. The first panel shows a forward vs. side scatter plot of the isolated neutrophils and the second panel is a CD80 fluorescence vs. side scatter dot plot (blue) with overlaid hamster-APC isotype (red), showing neutrophils are CD80 + . The third panel shows a representative modified Wright’s-stained image of a cytospin smear of the isolated cells, which were primarily segmented neutrophils. The vacuolated cytoplasm in one neutrophil is likely an artifact of the isolation procedure (scale bar = 20 μm).
Article Snippet: CD5-FITC/CD21-PE/CD45-APC , CD5 : Bio-Rad Cat# MCA1037F, RRID:AB_322643; CD21 : BD Biosciences Cat# 555422; RRID:AB_395816 CD45 : Bio-Rad Cat# MCA1042APC, RRID:AB_324810.
Techniques: Labeling, Isolation, Modification, Staining, Cell Counting, Fluorescence