Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour

Abstract

Magnetic sorting of cells, based on microbead conjugated Background antibodies (Abs), employs positive as well as negative immunomagnetic separation methods, for isolation of a specific cell population. These microbeads are suggested to be nontoxic, biodegradable carriers conjugated to various antibodies. Isolation of cells through positive selection involves the attachment of antibody conjugated microbeads to the cells of interest, followed by their isolation in the presence of a strong magnetic field to obtain higher purity. Negative selection involves attachment of microbead conjugated antibodies to all other cell populations except the cells of interest, which remain untagged. In the present study, we compared the two methods for their effect on functional and immunophenotypic behavior of isolated CD14+ monocytes.: Peripheral blood mononuclear cells (PBMCs) were isolated from Methods blood collected from healthy volunteers by density gradient centrifugation. Human blood derived monocytes were isolated through positive selection and negative selection, making use of the appropriate monocyte isolation kit. Monocytes were then stimulated with lipopolysaccharide (LPS) and their activation and proliferation capacity were examined. The degradation or dissociation of cell-bound microbeads was also investigated.: We observed an impaired LPS sensitivity as well as poor activation Results and proliferation capacity upon stimulation by LPS in positively sorted CD14+ monocytes as compared to negatively sorted CD14+ monocytes. The attached microbeads did not degrade and remained attached to the cells even after 6 days of culture.: Our results suggest that positively sorted CD14+ cells exhibit Conclusions hampered functionality and may result in inaccurate analysis and observations in downstream applications. However, these cells can be used for immediate analytical procedures.

Enhanced monocyte migratory activity in the pathogenesis of structural remodeling in atrial fibrillation

“…In this study, we evaluated the functional differences of circulating monocytes, focusing particularly on their migratory activity, in AF patients who underwent catheter ablation with or without advanced LA structural remodeling gauged by echocardiography-measured LAD. In the monocyte isolation, we employed the magnet-based negative selection method to collect cells without any stimulation, since in the positive selection method the phenotype of monocytes may be altered by binding to CD14 antibody [15,16]. Moreover, we found that gene products and protein levels of CCR2 in isolated monocytes were higher in AF patients whose LAD was larger than 39 mm.…”

A protocol for rapid monocyte isolation and generation of singular human monocyte-derived dendritic cells

“…Positive selection might affect surface marker expression and cell function. Bhattacharjee and collaborators show that positive selection of CD14 + cells results in changes of cell functionality, activation and proliferation when compared to negative selection, and this difference could affect downstream applications [18] such as LPS stimuli or moDCs differentiation. The authors suggested that the temporary binding of CD14 by microbeads could affect cell activation, since this receptor is important in TLR response [18,19].…”

“…Bhattacharjee and collaborators show that positive selection of CD14 + cells results in changes of cell functionality, activation and proliferation when compared to negative selection, and this difference could affect downstream applications [18] such as LPS stimuli or moDCs differentiation. The authors suggested that the temporary binding of CD14 by microbeads could affect cell activation, since this receptor is important in TLR response [18,19]. Zhou and collaborators also observed that negative selection yields less pure cultures, but they also discussed that, in studies using moDCs a few lymphocyte contaminants were not critical as lymphocytes would not survive more than 1 week in the absence of interleukin 2 (IL-2) [20].…”

Generation and Analysis of Human and Murine Osteoclasts

“…This method results in high purity, with typically <5% contaminating cells. However, the procedure is quite stressful for the cells, and it is unclear if monocytes change their behavior due to the labeling (Bhattacharjee, Das, Mishra, Sahay, & Upadhyay, 2017). As an alternative, negative-isolation kits are available, and these are less stressful for cells.…”