Comparing methods for ex vivo characterization of human monocyte phenotypes and in vitro responses

• Histopaque or CPT isolation does not affect monocyte marker expression or responses.
• CD14+ monocyte isolation from PBMC results in a loss of CD14intCD16+ monocytes.
• Delayed isolation affects monocyte marker expression and cytokine production.
• CD14+ monocyte isolation from PBMC is not required to assess monocyte activation.

Monocytes are key innate effector cells and their phenotype and function may be a useful biomarker of disease state or therapeutic response. However, for such an assay to be clinically feasible it needs to be simple and reproducible, which this study aimed to address. Peripheral blood mononuclear cells (PBMC)2 isolated from whole blood using Histopaque-1077 or cell preparation tubes (CPT) showed no difference in the ex vivo monocyte activation marker expression or in vitro responses; however, a delayed isolation using CPT significantly altered ex vivo and in vitro phenotypes and responses. Furthermore, purification of monocytes using CD14+ microbeads resulted in a loss of CD14lowCD16+ monocytes compared to PBMC samples. Thus, the use of CPT reduced complexity and time compared to Histopaque, and PBMC isolation allowed the analysis of all 3 major monocyte subsets. Finally, because the delayed isolation of PBMC from CPT significantly altered monocytes, time delays should be standardized.

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