Primate Monocytes - CD14, CD16 - Ziegler-Heitbrock

OMIP 083: A 21-marker 18-color flow cytometry panel for in-depth phenotyping of human peripheral monocytes.

Abstract

This 21-marker, 18-color panel was developed to accurately delineate monocyte subsets by manual gating and, once successfully gated, to characterize monocyte function in-depth. This panel was optimized on human peripheral blood mononuclear cells (PBMCs) density-separated from whole blood drawn into Cyto-Chex blood collection tubes (BCTs; Streck, La Vista, NE) from healthy adults. All samples were analyzed on a three-laser (violet-blue-red) Aurora full spectrum flow cytometer (Cytek Biosciences, Fremont, CA). Deep phenotyping was achieved by minimizing the number of channels used to exclude nonmonocyte cells and maximizing the number of markers included to monitor monocyte immune status. This panel was specifically designed to interrogate cell-surface expression of monocyte-centric antigens to reduce handling time and complexity, which are desirable characteristics of a clinically applicable protocol. Although not tested on cryopreserved PBMCs or with alternative BCTs, we anticipate that this panel could be used on these sample types with minimal optimization. For optimal staining, we recommend the use of density-separated PBMCs over red blood cell (RBC)-lysed whole blood. In supplementary material, we highlight our step-wise troubleshooting to achieve optimal staining of this panel.