Ms separation column

Manufactured by Miltenyi Biotec

Sourced in Germany

MS separation columns are designed for the isolation and purification of target cells or molecules from heterogeneous samples. They utilize a magnetic separation process to efficiently capture and separate the desired components from the sample.

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MS columns (268 citations) Separation columns (13 citations) MS MACS separation column (6 citations) MACS separation MS columns (5 citations) M‐Columns (2 citations) MS+ MiniMACS separation columns (2 citations) MACS Cell Separation MS Columns (2 citations)

15 protocols using ms separation column

Isolation and Characterization of CD4+ T Cell Subsets

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CD4+CD25− cells and CD4+CD25+ cells (hereinafter T_regs) were isolated from the PBMC population with the help of immunomagnetic beads as previously described^{14 (link)}. CD4+CD25− cells were further analyzed and purified using CD7 expression. In short, CD4+CD25− cells were treated with anti-CD7 (PE-conjugated) for 30 min followed by incubation with anti-PE antibody-conjugated microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) for 15 min (4 °C). Then, the resulting CD4+CD25−CD7+ cells and CD4+CD25−CD7− cells were purified by positive and negative selection, respectively, with MS separation columns (Miltenyi Biotec). CD4+CD25−CD7+ cells were further analyzed and sorted according to their levels of CD43 and CD45 expression (i.e., CD43_low, CD43_high, CD45_low, and CD45_high).
Similarly, CD4+CD25+ cells were treated with anti-Gal1 (PE-conjugated) for 30 min followed by incubation with anti-PE antibody-conjugated microbeads (Miltenyi Biotec) for 15 min (4 °C). Then, the resulting CD4+CD25+Gal1+ cells and CD4+CD25+Gal1− cells were purified by positive and negative selection, respectively, with MS separation columns (Miltenyi Biotec) and were further analyzed and sorted according to their levels of CD25 expression (i.e., CD25_low, CD25_med, and CD25_high).

Wei S., Cao D., Liu Z., Li J., Wu H., Gong J., Liu Y, & Wu Y. (2018). Dysfunctional immunoregulation in human liver allograft rejection associated with compromised galectin-1/CD7 pathway function. Cell Death & Disease, 9(3), 293.

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Assessing Dendritic Cell Activation

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CD14⁺ cells were enriched from freshly isolated PBMC with human CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and MS separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. DC were generated by cultivation of CD14⁺ cells with 800 IU/ml GM-CSF (PeproTech EC Ltd, London, United Kingdom) and 1000 IU/ml IL-4 (Miltenyi Biotec, Bergisch Gladbach, Germany) for 5 days. After 5 days, DC were incubated with increasing concentrations of abacavir (3, 10, 30 µg/ml) or NiSO4 (250 mM). After 24 h of co-incubation, DC were harvested, and the expression of co-stimulatory molecules (CD80, CD83, CD86 and CD40 (Anti-bodies from BD Biosciences, Basel, Switzerland)) was analyzed by flow cytometry. Furthermore, culture supernatants were analyzed for IL-1β, TNFα and IL-6 cytokine secretion by multiplex analysis (Meso Scal Discovery, Gaithersburg, MD USA).

Adam J., Wuillemin N., Watkins S., Jamin H., Eriksson K.K., Villiger P., Fontana S., Pichler W.J, & Yerly D. (2014). Abacavir Induced T Cell Reactivity from Drug Naïve Individuals Shares Features of Allo-Immune Responses. PLoS ONE, 9(4), e95339.

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Isolation of Murine T Cell Subsets

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T cells were purified from mouse spleens and lymph nodes (LN) as described (5 (link), 20 (link)). Briefly, single cell suspensions were incubated with purified anti-CD11b, -CD8, -B220, anti-Gr-1, -CD16/32, -Ter119 (BD Bioscience, San Jose, CA) and -I-A/E (eBioscience) mAbs. The cells were then washed and incubated with anti-rat dynabeads (Dynal, Invitrogen) according to the manufacturer’s instructions. Bulk CD4⁺ T cells were then purified by magnetic negative selection of the unwanted cells. For Treg isolation, these bulk CD4⁺ T cells were incubated with anti-CD25-PE mAb and CD4⁺CD25⁺ T cells isolated by positive selection using anti-PE microbeads and MS separation columns (Miltenyi Biotec). Purity was assessed by cytofluorometric analysis and was consistently 90–95%. The remaining cells were used as CD4⁺CD25⁻ Tconv.

Chen L.C., Nicholson Y.T., Rosborough B.R., Thomson A.W, & Raimondi G. (2016). A novel mTORC1-dependent, Akt-independent pathway differentiates the gut tropism of regulatory and conventional CD4 T cells. Journal of immunology (Baltimore, Md. : 1950), 197(4), 1137-1147.

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ADRC Subpopulation Enrichment by MACS

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Enrichment of CD31 positive and -negative ADRC subpopulations was accomplished by magnetic cell sorting (MACS) using MS separation columns (Miltenyi Biotech) under RNase-free conditions. Initially, the isolated ADRC was subjected to red cell lysis (RBC buffer, Miltenyi Biotech, cat no.130-094-183). Magnetic labelling was performed using a CD31 microbead kit (Miltenyi Biotech) after which cells were sorted on an OctoMACS™ Separator (Miltenyi Biotech). Hereby, CD31+ and CD31− ADRCs were obtained based on a positive or negative selection for the CD31 marker.

Dhumale P., Nielsen J.V., Hansen A.C., Burton M., Beck H.C., Jørgensen M.G., Toyserkani N.M., Haahr M.K., Hansen S.T., Lund L., Thomassen M., Sørensen J.A., Andersen D.C., Jensen C.H, & Sheikh S.P. (2023). CD31 defines a subpopulation of human adipose-derived regenerative cells with potent angiogenic effects. Scientific Reports, 13, 14401.

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Isolation of Endothelial Cells from Fibrin Hydrogels

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Cell samples from each group were isolated from fibrin hydrogels after 48h coculture by dissolution in 1 mg/mL collagenase (Sigma) for 30 min at 37°C and a cell pellet was formed by centrifugation and washed with PBS three times. HUVECs were positively selected for CD31 by magnetic cell sorting (MACS) using MS separation columns (Miltenyi Biotech, Bergisch Gladbach, Germany). Following instructions provided by the manufacturer, a positive selection for ECs using the CD31 MicroBead Kit (Miltenyi) was performed. PBS was used for the final washing step and elution from the column. The CD31⁺ population (HUVECs) was used in subsequent assays of endothelial function.

Piard C., Jeyaram A., Liu Y., Caccamese J., Jay S.M., Chen Y, & Fisher J. (2019). 3D printed HUVECs/MSCs cocultures impact cellular interactions and angiogenesis depending on cell-cell distance.. Biomaterials, 222, 119423.

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Isolation of Human Blood Monocytes

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Source material for all human blood cells was freshly collected buffy
coats provided by the Oklahoma Blood Institute. Blood donors were in good health
and negative for all infections tested for during standard donation. Age and
smoking history were not available on blood donors. PBMC were immediately
isolated by gradient centrifugation using Lymphoprep (STEMCELL TECNHOLOGIES)
according to manufacturer’s instructions. Resulting PBMC were either
resuspended in 1X PBS for subsequent staining and flow cytometry, or in 1X PBS
containing 0.5% BSA and 2 mM EDTA for monocyte enrichment. Positive
selection of monocytes was conducted using anti-CD14 MicroBeads along with MS
Separation Columns (Miltenyi Biotec) following manufacturer’s
instructions.

Patel V.I., Booth J.L., Duggan E.S., Cate S., White V.L., Hutchings D., Kovats S., Burian D.M., Dozmorov M, & Metcalf J.P. (2016). Transcriptional Classification and Functional Characterization of Human Airway Macrophage and Dendritic Cell Subsets. Journal of immunology (Baltimore, Md. : 1950), 198(3), 1183-1201.

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Murine CD8+ T-cell Purification

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Primary lymphocytes were isolated from Balb/c mouse spleens and activated by conA and IL-2 in vitro as previously described. Murine CD8α (Ly-2) MicroBeads (Miltenyi, Cat# 130-117-044), a MiniMACS separator (Miltenyi, Cat# 130-042-102), MS separation columns (Miltenyi, Cat# 130-402-201) and MACS MultiStand (Miltenyi, Cat# 130-402-303) were used for CD8⁺ T-cell purification. MACS was performed according to the Miltenyi MACS protocol; 95 µL MACS buffer was used for resuspension, and 10 µL microbeads were used for magnetic labeling every 1×10⁷ cells at 4°C for 15 min. CD8⁺ T cells and non-CD8⁺ T cells were then separated after MACS and counted for transfer. Flow cytometric analysis was used to confirm that the CD8⁺ T-cell population purity was over 90% (BD, LSRFortessa).

Quan Y., He J., Zou Q., Zhang L., Sun Q., Huang H., Li W., Xie K, & Wei F. (2023). Low molecular weight heparin synergistically enhances the efficacy of adoptive and anti-PD-1-based immunotherapy by increasing lymphocyte infiltration in colorectal cancer. Journal for Immunotherapy of Cancer, 11(8), e007080.

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Isolation of Immune Cells from OPSCC

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Fresh OPSCC tumors and matched adjacent normal tissue samples were placed on ice following surgical resection in Dulbecco's modified eagle medium (DMEM, USA), supplemented with 1% Penicillin/Streptomycin solution (Hyclone, USA). Subsequently, all samples were gently cut into small pieces on ice and digested using a tumor dissociation kit (Miltenyi Biotec, Germany), following the built-in program 37C_h_TDK_3 for tumor tissues and program 37C_h_TDK_1 for adjacent normal tissues. After disruption, the cells were filterd through a 40 mm filter (BD Biosciences, USA) and washed with 1x Hank's balanced salt solution (HBSS, USA). The resulting single-cell suspension was centrifuged at 500G for 5 minutes and resuspended in 4% bovine serum albumin (BSA, USA). Single-cell suspensions were then stained with CD3 MicroBeads (Miltenyi Biotec, Germany) and sorted into immune (CD3⁺) cells via magnetic separation using MS Separation columns (Miltenyi Biotec, Germany). Cell number and viability were checked after staining with 0.4% trypan blue staining (cell number > 5 × 10⁵ and viability > 80%).

Rao Y., Qiu K., Song Y., Mao M., Feng L., Cheng D., Li J., Zhang Z., Zhang Y., Shao X., Pang W., Wang Y., Chen X., Jiang C., Wu S., Yu S., Liu J., Wang H., Peng X., Yang L., Chen L., Mu X., Zheng Y., Xu W., Liu G., Chen F., Yu H., Zhao Y, & Ren J. (2024). The diversity of inhibitory receptor co-expression patterns of exhausted CD8+ T cells in oropharyngeal carcinoma. iScience, 27(5), 109668.

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Isolation and Culture of Mouse Monocytes and Macrophages

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Male C57BL/6 mice, 8–10 weeks old (Charles River Laboratories), were used in all experiments for the generation of monocytes and Mϕ. The animals were culled by CO₂ euthanasia immediately before the start of the bone marrow isolation procedure. Mouse bone marrow was harvested as previously described.³² (link) Briefly bone marrow was collected by flushing the hind-limb tibia and femur with DMEM (Gibco) containing 10% FBS (Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco). Cell suspension was filtered with a 70-μm cell strainer resuspended in 5 mL cold 1× red blood cell lysis buffer (BioLegend) before washing in PBS and performing gradient centrifugation using Histopaque-1077 (Sigma-Aldrich) and at 400 × g without brake for 30 min. The interphase was carefully collected as bone marrow mononuclear cells (BMMNCs). For monocyte collection, collected BMMNCs were subjected to magnetic sorting with the Monocyte Isolation Kit (Miltenyi Biotec) and MS Separation Columns (Miltenyi Biotec). For Mϕ production, isolated BMMNCs were plated at a density of 4 × 10³ cells/cm² and cultured for 6 days in complete DMEM containing mouse macrophage colony stimulating factor (M-CSF; 20 ng/mL; PeproTech) to differentiate to Mϕ.³² (link)

Fields L., Ito T., Kobayashi K., Ichihara Y., Podaru M.N., Hussain M., Yamashita K., Machado V., Lewis-McDougall F, & Suzuki K. (2021). Epicardial placement of human MSC-loaded fibrin sealant films for heart failure: Preclinical efficacy and mechanistic data. Molecular Therapy, 29(8), 2554-2570.

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Isolation and Characterization of MØ Subsets

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Cells were plated in hydrophobic-coated dishes (Sarstedt) or Ultra-Low attachment dishes (Corning) and detached by pipetting up and down after 30 min of incubation in PBS/5 mM EDTA/1% BSA. Next, HSApos and HSAneg MØ were isolated using biotinylated anti-HSA (clone M1/69; eBiosciences) and anti-Biotin Magnetic Beads (Miltenyi) in a MS separation column (Miltenyi). Cells were blocked with PBS/20% Normal Goat Serum (NGS)/10% human AB serum/1% BSA/5 mM EDTA and next labelled with PE-conjugated anti-HSA, APC-conjugated anti-HSA, APC-conjugated anti-CD4 (eBiosciences), or FITC-conjugated anti-p24 mAb (clone KC57, Beckman Coulter).

Deshiere A., Joly-Beauparlant C., Breton Y., Ouellet M., Raymond F., Lodge R., Barat C., Roy M.A., Corbeil J, & Tremblay M.J. (2017). Global Mapping of the Macrophage-HIV-1 Transcriptome Reveals that Productive Infection Induces Remodeling of Host Cell DNA and Chromatin. Scientific Reports, 7, 5238.

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