An Innovative Cascade System for Simultaneous Separation of Multiple Cell Types

Abstract

Isolation of different cell types from one sample by fluorescence activated cell sorting is standard but expensive and time consuming. Magnetic separation is more cost effective and faster by but requires substantial effort. An innovative pluriBead-cascade cell isolation system (pluriSelect GmbH, Leipzig, Germany) simultaneously separates two or more different cell types. It is based on antibody-mediated binding of cells to beads of different size and their isolation with sieves of different mesh-size. For the first time, we validated the pluriSelect system for simultaneous separation of CD4+- and CD8+-cells from human EDTA-blood samples. Results were compared with those obtained by magnetic activated cell sorting (MACS; two steps -first isolation of CD4+, then restaining of the residual cell suspension with anti-human CD8+ MACS antibody followed by the second isolation). pluriSelect separation was done in whole blood, MACS separation on density gradient isolated mononuclear cells. Isolated and residual cells were immunophenotyped by 7-color 9-marker panel (CD3; CD16/56; CD4; CD8; CD14; CD19; CD45; HLA-DR) using flow cytometry. Cell count, purity, yield and viability (7-AAD exclusion) were determined. There were no significant differences between both systems regarding purity (MACS (median[range]: 92.4% [91.5-94.9] vs. pluriSelect 95% [94.9-96.8])) of CD4+ cells, however CD8+ isolation showed lower purity by MACS (74.8% [67.6-77.9], pluriSelect 89.9% [89.0-95.7]). Yield was not significantly different for CD4 (MACS 58.5% [54.1-67.5], pluriSelect 67.9% [56.8-69.8]) and for CD8 (MACS 57.2% [41.3-72.0], pluriSelect 67.2% [60.0-78.5]). Viability was slightly higher with MACS for CD4+ (98.4% [97.8-99.0], pluriSelect 94.1% [92.1-95.2]) and for CD8+-cells (98.8% [98.3-99.1], pluriSelect 86.7% [84.2-89.9]). pluriSelect separation was substantially faster than MACS (1h vs. 2.5h) and no pre-enrichment steps were necessary. In conclusion, pluriSelect is a fast, simple and gentle system for efficient simultaneous separation of two and more cell subpopulation directly from whole blood and provides a simple alternative to magnetic separation.

Time course of chemotaxis and chemokinesis of neutrophils following stimulation with IL-8 or FMLP

“…Whereas the microbead method enables isolation of purified PMNs in sufficient quantity, viability and without pre-activation, the 3D-µ-slide facilitates high-quality microscopy, linear concentration gradients and support of long-term assays. [7][8][9]…”

Up-modulation of PLC-β2 reduces the number and malignancy of triple-negative breast tumor cells with a CD133+/EpCAM+ phenotype: a promising target for preventing progression of TNBC

“…In order to study the correlation of CD133 and/or EpCAM with malignant features of MDA-MB-231, a magnetic step-by-step cell isolation with antibodies directed against the two surface antigens was performed. Since CD133 + cells are rare elements in the MDA-MB-231 cell population, we applied the MACS technique instead of the currently used Fluorescence-Activated Cell Sorting, thus ensuring the achievement of a relatively high number of cells in a short time [ 17 , 23 ]. We obtained populations enriched in CD133 − /EpCAM − , CD133 − /EpCAM + , CD133 + /EpCAM − or CD133 + /EpCAM + cells (Fig.…”

“…MDA-MB-231 sub-populations enriched in CD133 + and/or EpCAM + cells were obtained by means of the MACS immunomagnetic separation system, essentially as described by Pierzchalski et al [ 23 ]. In particular, cells were firstly labeled with CD133/1 MicroBeads (Miltenyi Biotech) and subjected to positive magnetic cell separation through MACS SD columns in the field of the MACS magnet (Miltenyi Biotech), according to manufacturer’s instructions.…”

Stem cell separation from cord blood

“…A weak point in the study is that we did not test viability of the harvested stem cells. Pierzchalski et al (10) reported that high cell numbers can be isolated by this method. Positive selection, by labeling the target cells, is the fastest and the most efficient way to isolate a cell subset with high purity and Stem cell separation from cord blood @ C I C E d i z i o n i I n t e r n a z i o n a l i yield, but because separation is based on a single parameter (i.e., magnetization), this method is generally effective only for the isolation of a single cell population.…”