Ls separation column

Manufactured by Miltenyi Biotec

Sourced in Germany, United States, United Kingdom

The LS separation columns from Miltenyi Biotec are designed for the magnetic separation of cells. They are used in conjunction with a MACS Separator to efficiently isolate target cells from complex samples.

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LS MACS cell separation columns LS magnetic separation columns LS columns of MACS cell separation system MACS Separation LS columns MACS magnetic LS separation columns Magnetic separation LS column LS cell separation column MACS LS separation columns LS columns Separation columns

23 protocols using ls separation column

Isolation of CD34+ Progenitor Cells

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CD34⁺ progenitors were isolated by immunomagnetic bead selection on an
affinity column using magneticactivated cell-sorting (MACS) CD34 Isolation Kit (130046702,
Miltenyi Biotec, Bergisch-Gladbach, Germany). Briefly, the MNCs were suspended in PBS (up
to 108 total cells). Then, 100 µl human Fc receptor (FcR) blocking reagent and 100 µl
anti-CD34 microbeads were added, mixed and incubated at 4°C for 30 minutes. Subsequently,
the MNCs were washed with PBS and centrifuged at 300 g for 10 minutes. The labeled MNCs
were then re-suspended in 3 mL PBS and passed through an LS separation column (130042401,
Miltenyi Biotec, Bergisch-Gladbach, Germany) placed in a magnetic field to capture the
CD34⁺ cells. In order to collect the CD34⁺ cells, the column was
removed from the magnetic field and flushed with a PBS and 2 mM EDTA washing solution. The
purity of CD34⁺ cells was assessed by flow cytometry using antihuman
phycoerythrin (PE) -conjugated CD34 antibody (550761, Pharmingen, San Diego, CA, USA).

Naseri Mobaraki S., Abroun S., Atashi A, & Kaviani S. (2023). Evaluation of Expansion and Maintenance of Umbilical Cord Blood CD34+ Cells in The Co-Culture with Umbilical Cord Blood-Derived Mesenchymal Stem Cells in The Presence of Microcarrier Beads. Cell Journal (Yakhteh), 25(3), 184-193.

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Microglia Isolation and Purity Analysis

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Single-cell suspension was first incubated in Fc receptor blocking buffer (Miltenyi Biotec, 130-092-575) for 10 min to minimize microglia activation during handling and then incubated with anti-CD11b-MicroBeads (Miltenyi, 130-093-634) for 10 min at 4 °C in the dark. The sample then went through the LS separation column (Miltenyi Biotec, 130-042-401) for positive selection. Finally, an aliquot of purified cells was further incubated with an anti-CD11b-FITC monoclonal antibody (M1/70) (eBioscience, 11-0112-81) for 30 min at 4 °C in the dark, and the purity of CD11b + cells was analyzed using Beckman flow cytometer.

Zhang Y., Dong Y., Zhu Y., Sun D., Wang S., Weng J., Zhu Y., Peng W., Yu B, & Jiang Y. (2022). Microglia-specific transcriptional repression of interferon-regulated genes after prolonged stress in mice. Neurobiology of Stress, 21, 100495.

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Isolation of Murine Erythroblasts

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Splenic cell suspension was incubated with 2 mL red blood cell (RBC) lysis buffer (R1010, Solarbio, Beijing, China) on ice for 5 min to remove mature RBCs. Isolation of erythroblasts was performed according to the manufacturer’s recommendations (130-049-901, Miltenyi Biotec, Cologne, Germany). Briefly, cells were incubated with 10 µL anti-Ter119 MicroBeads (130-049-901, Miltenyi Biotec, Cologne, Germany) per 10⁷ cells in 100 μL PBS containing 2% fetal bovine serum (FBS) for 20 min at 4 °C. Cells were resuspended in 500 µL PBS after washing, and loaded into the LS separation column (130-042-401, Miltenyi Biotec). The column was removed from the separator after washing with PBS and the Ter119-labeled cells were flushed out from the column. The Ter119⁺ cell fraction was collected for RNA extraction or preparation for protein lysate.

Yang L., He S., Ling L., Wang F., Xu L., Fang L., Wu F., Zhou S., Yang F., Wei H, & Yu D. (2023). Crosstalk between miR-144/451 and Nrf2 during Recovery from Acute Hemolytic Anemia. Genes, 14(5), 1011.

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Magnetic Separation of BCSC Subsets

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After the preparation of single-cell suspension, magnetic separation of the BCSCs subsets was done using the LS separation column (Miltenyi Biotec B.V. & Co. KG) according to the manufacturers' instructions. The cells were separated by magnetic selection after staining with CD24, CD44 and CD326 (EpCAM) Micro Beads labeled with monoclonal antibodies. Finally, different subsets of cells were collected by magnetic separation and stored for subsequent RNA extraction. Accordingly, the cells were divided into three groups including G1: CD44⁺/CD24⁻/EpCAM⁻ cells; G2: CD44⁺/CD24^−//EpCAM⁺ cells; and G3: mammospheres,

Zekri A.R., Bahnassy A., Mourad M., Malash I., Ahmed O, & Abdellateif M.S. (2022). Genetic profiling of different phenotypic subsets of breast cancer stem cells (BCSCs) in breast cancer patients. Cancer Cell International, 22, 423.

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Isolation of Murine PMN-MDSCs

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Isolation of CD11b⁺Ly6G⁺ PMN-MDSCs was performed using a mouse MDSC isolation kit (Miltenyi Biotec, FL) according to the manufacturer’s instructions. Briefly, after FcR blocking, cells were stained by anti–Ly6G–biotin antibody and labeled with antibiotin microbeads. The cell suspension was passed through an LS separation column (Miltenyi Biotec) and the retained cells were the purified murine MDSCs (CD11b⁺Gr-1^hiLy6G⁺ population).

Zhang F., Hu K., Liu W., Quan B., Li M., Lu S., Chen R., Ren Z, & Yin X. (2022). Oxaliplatin-Resistant Hepatocellular Carcinoma Drives Immune Evasion Through PD-L1 Up-Regulation and PMN-Singular Recruitment. Cellular and Molecular Gastroenterology and Hepatology, 15(3), 573-591.

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Isolation and Culture of Human NK Cells

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Human peripheral blood mononuclear cells (PBMCs) were isolated from human buffy coats from the New York Blood Center by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Primary NK cells were extracted from PBMCs by negative immunomagnetic isolation using a NK cell isolation kit (Miltenyi Biotec) according to the manufacturer's protocol. Briefly, cells were incubated with NK Cell Biotin-Antibody Cocktail and NK Cell MicroBead Cocktail to label non-NK cells, followed by loading onto a LS Separation column (Miltenyi Biotec). The cell suspension was then placed in the magnetic field of a MACS Separator to separate labeled from non-labeled cells. The isolated human NK cells were subsequently cultured in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% fetal bovine serum, 1x penicillin/streptomycin and 100 U/mL IL-2 at 37°C in 5% CO 2 . The purity of the NK cell population was validated by flow cytometry using PE-Vio 770 anti-human CD56 (1:50; REA196 clone; Miltenyi Biotec) and FITC anti-human CD3 (1:50; REA613 clone; Miltenyi Biotec) antibodies.

Park S., Choi S., Shimpi A.A., Estroff L.A., Fischbach C, & Paszek M.J. (2024). Collagen Mineralization Decreases NK Cell-Mediated Cytotoxicity of Breast Cancer Cells via Increased Glycocalyx Thickness. Advanced materials (Deerfield Beach, Fla.).

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Isolation and Culture of Primary Human NK Cells

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Human peripheral blood mononuclear cells (PBMCs) were isolated from human buffy coats from the New York Blood Center by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Primary NK cells were extracted from PBMCs by negative immunomagnetic isolation using a NK cell isolation kit (Miltenyi Biotec) according to the manufacturer’s protocol. Briefly, cells were incubated with NK Cell Biotin-Antibody Cocktail and NK Cell MicroBead Cocktail to label non-NK cells, followed by loading onto a LS Separation column (Miltenyi Biotec). The cell suspension was then placed in the magnetic field of a MACS Separator to separate labeled from non-labeled cells. The isolated human NK cells were subsequently cultured in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% fetal bovine serum, 1x penicillin/streptomycin and 100 U/mL IL-2 at 37°C in 5% CO₂. The purity of the NK cell population was validated by flow cytometry using PE-Vio 770 anti-human CD56 (1:50; REA196 clone; Miltenyi Biotec) and FITC anti-human CD3 (1:50; REA613 clone; Miltenyi Biotec) antibodies.

Park S., Choi S., Shimpi A.A., Estroff L.A., Fischbach C, & Paszek M.J. (2024). COLLAGEN MINERALIZATION DECREASES NK CELL-MEDIATED CYTOTOXICITY OF BREAST CANCER CELLS VIA INCREASED GLYCOCALYX THICKNESS. bioRxiv.

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Microglia Isolation from Mixed Glia

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We gently scraped and applied the cells to an antigen–antibody-mediated magnetic cell-sorting (MACS, Miltenyi Biotec) assay to positively select microglia. Briefly, the mixed glial population was re-suspended in MACS buffer (Miltenyi Biotec) and incubated with CD11b MicroBeads (Miltenyi Biotec). The cell suspension was then applied to LS separation column (Miltenyi Biotec) fitted into a QuadroMACS cell separator (Miltenyi Biotec). Unlabeled cells were allowed to pass through the column while labeled cells remained captured in the magnetic field. After washing the column with MACS buffer, the column was then removed from the magnetic separator and flushed with MACS buffer to collect the purified microglia population. For an increased level of purity, the eluted microglia population was passed through a new LS separation column a second time. The purity of microglia used in our study was more than 95% assessed by immunocytochemistry (data not shown). Microglia either acutely collected from the LS separation column or incubated on a poly-l-lysine-coated plate for 24 h were homogenized, and total RNA was extracted using RNeasy Plus Mini Kit (QIAGEN).

He Y., Yao X., Taylor N., Bai Y., Lovenberg T, & Bhattacharya A. (2018). RNA sequencing analysis reveals quiescent microglia isolation methods from postnatal mouse brains and limitations of BV2 cells. Journal of Neuroinflammation, 15, 153.

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Isolation of Adult Mouse Microglia

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Adult microglia were isolated using a neural dissociation kit (Miltenyi Biotec) followed by magnetic-activated cell sorting with CD45 micro beads (Miltenyi Biotec) according to the manufacturer’s instructions. CD45 antibody–coupled beads were used as a replacement for CD11b beads, as the receptor is lacking in the CR3-deficient animals. In brief, perfused brains of 3–4-mo-old mice were dissociated using the neuronal dissociation kit (Miltenyi Biotec), incubated with CD45 microbeads (Miltenyi Biotec), and separated using an LS separation column (Miltenyi Biotec). For functional studies, the cells were allowed to recover for 3 d in DMEM/F12 with 10% FBS. Cells for RNA were immediately frozen at −80°C. Cells used for flow cytometry were kept on ice and stained immediately.

Czirr E., Castello N.A., Mosher K.I., Castellano J.M., Hinkson I.V., Lucin K.M., Baeza-Raja B., Ryu J.K., Li L., Farina S.N., Belichenko N.P., Longo F.M., Akassoglou K., Britschgi M., Cirrito J.R, & Wyss-Coray T. (2017). Microglial complement receptor 3 regulates brain Aβ levels through secreted proteolytic activity. The Journal of Experimental Medicine, 214(4), 1081-1092.

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Isolation and Differentiation of Human Macrophages

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Human peripheral blood mononuclear cells (PBMCs) were obtained from leukopaks. Briefly, whole blood from leukopaks was mixed with 1× PBS, and Ficoll-Hypaque solution (Cytiva) was carefully laid underneath of the blood and PBS mixture. Cells were centrifuged at 400g for 30 min with no break. After the centrifugation, cells in the buffy coat were collected and lysed with ACK lysis buffer (Gibco) on ice for 5 min and washed with excess amounts of RPMI (Gibco) to obtain PBMCs. CD14⁺ monocytes were isolated (>95% purity; Extended Data Fig. 4g) from the PBMCs by using CD14 MicroBeads (Miltenyi Biotec) following the manufacturer’s instructions. One hundred million cells were incubated with 200 μl of CD14 MicroBeads for 15 min at 4 °C, washed with MACS buffer and loaded onto an LS separation column (Miltenyi Biotec). CD14⁺ cells were eluted from the column after three washes with MACS buffer. The purity of the monocytes was evaluated via flow cytometry.
Human macrophages were differentiated from the monocytes. Briefly, CD14⁺ monocytes were cultured in RPMI-1640 supplemented with 10% FBS, 100 U ml⁻¹ pen–strep and 50 ng ml⁻¹ human macrophage colony-stimulating factor (M-CSF; BioLegend) at 37 °C with 5% CO₂ for 7 d. Medium was refreshed on day 4, and before signaling assays, human macrophages were starved in growth medium overnight with 2% FBS.

Krüger S., Brandt E., Klinger M., Krüger S, & Kreft B. (2000). Interleukin-8 Secretion of Cortical Tubular Epithelial Cells Is Directed to the Basolateral Environment and Is Not Enhanced by Apical Exposure to Escherichia coli. Infection and Immunity, 68(1), 328-334.

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