Cells were sorted for the cell surface marker CD56 using immunomagnetic column sorting (MACS) to achieve pure populations of muscle precursor cells, as described by Agley et al. (2015) (link). Cells grown to ∼50% confluency in a 10 cm culture dish were incubated with Human CD56 primary antibody conjugated magnetic microbeads (Miltenyi Biotec) at 4°C for 30 min. CD56+ myoblasts were filtered from the bulk population using a magnetic cell separator (Miltenyi Biotec) according to the manufacturer’s instructions (Miltenyi Biotec).
Magnetic cell separator
Manufactured by Miltenyi Biotec
Sourced in Germany
The Magnetic cell separator is a laboratory instrument used for the separation and enrichment of target cells from heterogeneous cell populations. It utilizes magnetic beads coated with specific antibodies or ligands to bind to the cells of interest, which are then separated from the rest of the sample using a magnetic field.
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Lab products found in correlation
X vivo 15 serum free medium, by Lonza (1 mentions)
Cd14 microbead, by Miltenyi Biotec (1 mentions)
Lymphocyte separation medium, by GE Healthcare (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Anti sca 1 immunomagnetic microbeads, by Miltenyi Biotec (1 mentions)
Ethylene diamine tetraacetic acid (edta), by ICN Biomedicals (1 mentions)
Pacific blue conjugated anti cd4, by BioLegend (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Facscan, by BD (1 mentions)
7 protocols using magnetic cell separator
1
Isolating Muscle Precursor Cells via CD56 Sorting
2
Allogenic NK Cell Preparation for HIV Treatment
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Allogenic NK cells were prepared under good manufacturing practice (GMP) conditions in a certified laboratory.[6 (link)] The donors were selected based on DNA genotyping of their human leukocyte antigen (HLA)-Cw mismatch with the killer cell immunoglobulin-like receptor (KIR) of HIV-1 patients, and their peripheral blood mononuclear cells (PBMC) were collected. KIR/HLA-Cw mismatch was defined as the absence of one or more HLA alleles known to be ligands for the inhibitory KIR typing, based on previously published criteria.[7 (link)] The human NK cells in vitro culture synergistic kit (HAHK Bioengineering Co. Ltd., Shenzhen, China) was used according to the manufacturer's instructions, with X-VIVO 15 serum-free medium (Lonza, Walkersville, MD, USA), interleukin (IL)-2, and membrane chimeric cellular factors including IL-21 which co-stimulate expansion and activation of NK cells in the PBMC for about 14 days. The NK cells were then purified by a magnetic cell separator (Miltenyi Biotec, Germany). The final products were used for the infusion. The details of the method and cell product profile have already been published.[6 (link)]
3
Monocyte Isolation and CD137-Fc Culture
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Monocytes were isolated using CD14 microbeads and a magnetic cell separator (Miltenyi Biotech). Cells were cultured in a plate coated with CD137-Fc protein (10 μg/ml; R&D Systems) for 7 d as previously described (Kwajah and Schwarz, 2010 (link)).
4
Circulating Tumor Cell Isolation
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Anticoagulant citrate dextrose blood collection tubes, fluorescein isothiocyanate-labeled mouse anti-human cytokeratin monoclonal antibody, and phycoerythrin-labeled mouse anti-human CD45 monoclonal antibody were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). A magnetic bead-labeled anti-human EpCAM monoclonal antibody, a MACS® magnetic bead LS separation column, and a magnetic cell separator were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Lymphocyte separation medium was purchased from GE Healthcare (Waukesha, WI, USA). The nuclear fluorescent dye, DAPI (4, 6-diamidino-2-phenylindole) was obtained from Sigma (St Louis, MO, USA). Poly-lysine-coated slides were purchased from Thermo Electron (Pittsburgh, PA, USA). A fluorescent microscope (Olympus, Tokyo, Japan) was used for observation.
5
Sca-1+ Vascular Progenitor Cell Isolation
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Vascular progenitor cells (VPCs) were sorted with anti‐Sca‐1 immunomagnetic microbeads (Miltenyi Biotec, GmbH, Bergisch Gladbach, Germany) and then selected using a magnetic cell separator (Miltenyi Biotec, GmbH, Bergisch Gladbach, Germany). Sca‐1+ VPC populations were expanded for up to 5 population doublings.
6
Isolation and Purification of CD4+ T Cells
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From each cultured splenocyte sample CD4+ T cells were isolated by positive selection using CD4 magnetic microbeads and a magnetic cell separator (Miltenyi Biotech) according to the manufacturer’s instructions. The purity of the CD4+ T cells was determined using flowcytometry. Briefly, the isolated cells were stained with Pacific blue-conjugated anti-CD4 (Biolegend) in FACS buffer (PBS (pH 7.2) supplemented with 0.5% BSA (Sigma Aldrich) and 0.5 mM EDTA (ICN Biomedicals). After washing, data were acquired on a FACS Canto II (BD Biosciences) and analyzed using FlowJo software (Tree Star). The purity of the isolated CD4+ T cells was >95%.
7
Isolation and Purification of CD4+ T Cells
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Heparinized human venous blood was obtained from five healthy subjects (all male, 41.0 ± 10.1 years) after obtaining their written informed consent, which was approved by the Ethics Committee of Showa University (Approved No. 190613; Date of approval: 1 June 2019). Peripheral-blood mononuclear cells (PBMCs) were then obtained after centrifugation (1000× g for 30 min) of blood with lymphocyte separation medium (Organon Technica, Durham, NJ, USA). CD4+ T cells were purified from PBMCs using a magnetic cell separator (Milteny Biotec, Bergisch Gladbach, Germany) as described previously [20 (link)]. The cells were suspended in RPMI-FBS at a concentration of 1 × 106 cells/mL. The cell purity was more than 95%, as judged using a flow cytometer (FACScan; Becton Dickinson, San Jose, CA, USA).
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