Evaluation of a 12‐color flow cytometry panel to study lymphocyte, monocyte, and dendritic cell subsets in humans

Abstract

Monitoring changes in human immune cell populations such as lymphocytes, monocytes, and dendritic cells (DCs) during infectious diseases like human immunodeficiency virus (HIV) is crucial. However, difficulties to identify rare or heterogeneous cell populations can be limiting. For example, to accurately measure DC subsets, eight flow cytometry parameters are ideal. The aim of this work was to analyze the phenotype of human lymphocyte, monocyte, and DC subsets using a single 12-color flow cytometry panel. After erythrocyte lysis, blood from healthy human volunteers was washed and labeled with a cocktail of 12 antibodies. Samples were analyzed on a Becton-Dickinson FACSAria TM equipped with three lasers. Data were compared with lineage-specific panels using 5-8 Ab combinations per lineage. Acquired data were analyzed using FlowJo software. Our 12-color panel allows for the identification of the following major subsets of circulating cells in a single tube: CD41 and CD81 T lymphocytes, B lymphocytes, NK cells, NKT cells, monocyte subsets (CD14 and/or CD16), and five nonoverlapping HLA-DR1Lin2 subsets: CD341 hematopoietic stem cells, CD1231 plasmacytoid DC, and three subsets of CD11c1 myeloid DC expressing either CD16, CD1c (BDCA-1), or CD141 (BDCA-3). We have developed a single flow cytometry panel that allows for simultaneous detection of the lymphocyte and monocyte cell populations and all known DC subsets. Studying these major players of the immune system in one single panel may give us a broader view of the immune response during HIV infection and the ability to better define the role of individual cell types in Acquired Immune Deficiency Syndrome (AIDS) pathogenesis. ' 2010 International Society for Advancement of Cytometry

Identifying leukocyte populations in fresh and cryopreserved sputum using flow cytometry

“…Although this study was limited to four fluorophores, FCM analysis of IS could be extended to identify several more important cell populations, such as DCs and lymphocyte subsets (e.g. 29,20), within a single antibody panel, as has been recently conducted with a 12-color panel in peripheral blood (30). Such an approach could be of considerable benefit for the detailed immunophenotyping of IS in respiratory diseases such as asthma.…”

Airway CD8+ T Cells Are Associated with Lung Injury during Infant Viral Respiratory Tract Infection

“…Cells were stained with innate cell-and T cell-specific antibody panels, as described previously (15,16), and analyzed using the LSRII flow cytometer (BD Biosciences, San Diego CA) and Flowjo software (Treestar, Ashland, OR).…”

Multi‐parametric phospho‐flow cytometry: A crucial tool for T lymphocyte signaling studies

“…The effector memory (CD45RA2CCR72) and TEMRA (CD45RA1CCR72) T lymphocytes lose expression of CD27 and CD28; cells with these phenotypes may be present at frequencies as low as 1% (6). This typical example is also true for other cell types such as dendritic cells (7) and may be applied to all leukocytes (8). It is expected that the number of identifiable subpopulations will increase with the advent of recently developed mass spectrometry-based cytometers (9).…”