Peripheral blood mononuclear cells (PBMC) were obtained and cultured from blood collected from healthy donors who voluntarily consented, as previously described (11 (link)). Briefly, cells were separated by a Ficoll-Paque® (GE Healthcare) density gradient centrifugation at 400 x g for 30 min at 4˚C. The monocytes-enriched layer was collected, plated for 2 h and non-adherent cells were removed with PBS. The pure monocytes were positively selected by an anti-CD14-coated microbead (MiniMACS separation column; Milteny Biotec, Inc.) as previously described (11 (link)). The cells were >95% viable, as assessed by the Trypan blue exclusion method, and consisted of >90% monocytes, as determined by a flow cytometry analysis. Flow cytometry (Coulter® Epics® XL-MCL™ flow cytometer including System II™ software; Beckman Coulter, Inc.) was based on CD14 and CD45 antigen expression after cell incubation for 30 min at 4˚C in PBS containing 2% FBS (Gibco®; Thermo Fisher Scientific, Inc.) with mouse monoclonal anti-CD14 (phycoerythrin-cyanin 5.5; clone RMO52; IgG2a; cat. no. A70204; Beckman Coulter, Inc.) and anti-CD45 (fluorescein-5-isothiocyanate; clone 30-F11; IgG2b; cat. no. 103107; BioLegend, Inc.) antibodies (data not shown).
Minimacs separation column
Manufactured by Miltenyi Biotec
Sourced in Germany
The MiniMACS separation column is a compact and versatile magnetic separation device designed for cell separation and purification. It utilizes a strong magnetic field to capture and hold magnetic labeled cells, allowing for the separation and enrichment of target cell populations. The MiniMACS separation column provides a simple and efficient means of performing cell isolation and purification, suitable for a wide range of applications in cell biology and biotechnology research.
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Lab products found in correlation
2 protocols using minimacs separation column
1
Isolation and Purification of Human Monocytes
2
Monocyte Isolation from Cancer Patients
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Peripheral blood mononuclear cells (PBMC) were isolated from citrated blood of cancer patients DVT+ and DVT- and healthy controls by Ficoll-Paque. For isolation of monocytes, PBMC were placed for 2 h in culture plate, and the non-adherent cells were removed with three changes of warm PBS. Pure monocytes were then positively selected by anti-CD14-coated magnetic microbeads (Mini MACS separation column; Milteny Biotec, Bergisch Gladbach, Germany) following manufacturer’s instructions. Monocyte purity was > 98%, as assessed by flow cytometry (data not shown). The cells were pooled and resuspended in a final concentration of 1x106 cells/ml in a freeze medium consisting of 30% autologous citrated plasma, 60% Iscove's and 10% DMSO. Next the cells were frozen in aliquots of 1 ml in sterile cryovials (Greiner, Germany) using a standard controlled freezing procedure and then stored in liquid nitrogen.
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