Macs separation kit

Manufactured by Miltenyi Biotec

Sourced in Germany

The MACS separation kit is a laboratory equipment designed for the magnetic separation of cells or biomolecules. It utilizes magnetic microbeads that can be conjugated to specific antibodies or ligands, allowing for the isolation and purification of target cells or molecules from complex biological samples.

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16 protocols using macs separation kit

CD3+ T cell Co-culture with BMMSCs

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CD3⁺ T cells were isolated by using MACS separation kit (Miltenyi, Auburn, CA, USA). CD3⁺T cells stained with carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen, Carlsbad, CA, USA) were co-cultured with BMMSCs. The method was detailed in our previous article [37 (link)].

Guo J., Fei C., Zhao Y., Zhao S., Zheng Q., Su J., Wu D., Li X, & Chang C. (2017). Lenalidomide restores the osteogenic differentiation of bone marrow mesenchymal stem cells from multiple myeloma patients via deactivating Notch signaling pathway. Oncotarget, 8(33), 55405-55421.

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Isolation and Analysis of Murine T Cells

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Single-cell suspensions were obtained from thymus and spleens of wild-type B6, CD4-cre NKAP cKO, Bax KO, Bax KO/CD4-cre NKAP cKO, Bcl-xL tg, Bcl-xL tg/CD4-cre NKAP cKO, Rag1-GFP WT, Rag1-GFP CD4-cre NKAP cKO, and bone marrow chimeras. For the complement experiments, freshly harvested splenocytes were incubated in GVB⁺⁺ buffer (Complement Technology) for 1 hour at room temperature prior FACS staining. For Annexin-V/DAPI staining, splenocytes were freshly harvested, resuspended in 1X binding buffer (eBioscience) and stained with fluorescence-conjugated Annexin-V for 30 minutes at 4°C, and cells stained with DAPI (invitrogen) in the last step. Stained cells were analyzed with a LSR II (BD Bioscience) flow cytometer and the data were analyzed with FlowJo 9.2 software (Tree Star). All data were doublet excluded with FSC-W/FSC-H and SSC-W/SSC-H before analysis, and dead cell were excluded using DAPI in all experiments except Annexin-V experiments. To obtain splenic CD4⁺Rag1-GFP⁺ T cells, non-CD4-expressing cells were eliminated by negative selection from splenic single-cell suspensions using a MACS separation kit (Miltenyi Biotec). Enriched CD4⁺ cells were stained and analyzed on a FACSAria cell sorter (BD Bioscience), and CD4⁺Rag1-GFP⁺ T cells were collected.

Hsu F.C., Shapiro M.J., Chen M.W., McWilliams D.C., Seaburg L.M., Tangen S.N, & Shapiro V.S. (2014). Immature recent thymic emigrants are eliminated by complement. Journal of immunology (Baltimore, Md. : 1950), 193(12), 6005-6015.

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Isolation and Co-culture of Naive T-cells with ccDC

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CD4⁺CD45RA⁺ naïve T-cells were isolated from PBMC by negative selection using MACS separation kit, following the manufacturer’s protocol (Miltenyi Biotec), and resuspended in IMDM medium supplemented with 10% FCS, 20 µg/mL apotransferrine (Sigma-Aldrich), 50 µM β-mercaptoethanol (Sigma-Aldrich), penicillin (100 U/mL) and streptomycin (100 µg/mL). Naïve T-cells (1 × 10⁶) were co-cultured with ccDC (0.1 × 10⁶) from IEC/moDC culture, after IEC/PBMC exposure, in 24 well flat-bottom plates for 5 to 6 days in the presence of 1 ng/mL TGF-β (Prospec) (Figure 1D). After incubation, the supernatant was collected and stored at −20 °C for cytokine analysis. After the ccDC/T-cell co-culture, over 90% of the CD4+ T-cell were viable. The viability was not affected by exposure to NDO and/or CpG.

Ayechu-Muruzabal V., Overbeek S.A., Kostadinova A.I., Stahl B., Garssen J., van’t Land B, & Willemsen L.E. (2020). Exposure of Intestinal Epithelial Cells to 2′-Fucosyllactose and CpG Enhances Galectin Release and Instructs Dendritic Cells to Drive Th1 and Regulatory-Type Immune Development. Biomolecules, 10(5), 784.

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Granulocyte-mediated T cell suppression

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Granulocytes (Ly-6G⁺) were purified from the lungs of mice that were administered with B16F10 TCM or from the spleens of MC38 tumor-bearing mice using MACS Separation kit (Miltenyi Biotec). 1×10⁵ isolated granulocytes cells were incubated with OT-1 WT splenocytes by different ratio 1:1, 1:2, 1:4. The mixed cells were co-cultured in 96 well plate with SIINFEKL (0.5 ng/μl) peptide stimulation for 48 hr, and then ³H thymidine (PerkinElmer) was added (1 μl/well for 24 hr). The radioactivity of samples was counted with a TopCount NXT instrument (PerkinElmer) as previously described^{59 (link)}.

Gui J., Zahedi F., Ortiz A., Cho C., Katlinski K.V., Alicea-Torres K., Li J., Todd L., Zhang H., Beiting D.P., Sander C., Kirkwood J.M., Snow B.E., Wakeham A.C., Mak T.W., Diehl J.A., Koumenis C., Ryeom S.W., Stanger B.Z., Puré E., Gabrilovich D.I, & Fuchs S.Y. (2020). Activation of p38α stress-activated protein kinase drives the formation of the pre-metastatic niche in the lungs. Nature cancer, 1(6), 603-619.

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Isolation and Characterization of CD271+ Bone Marrow Cells

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Density gradient centrifugation was performed to obtain bone marrow mononuclear cells (BMMNCs). CD271⁺ cells were selected using MACS separation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD271⁺-selected cells were cultured in 6-well plates at low density for 10 days and analyzed by flow cytometry for the expression of CD271 and alkaline phosphatase (ALP, TRA-2-49). The monoclonal antibodies are CD271-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) and TRA-2-49-PE (Biolegend, San Diego, CA, USA).

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In Vitro Treg Cell Differentiation and Coculture

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Mouse iTreg were differentiated from naive CD4⁺ T cells using the commercial CellXVivo Treg cell differentiation kits (CDK007, R&D). Briefly, naive CD4⁺ T cells were isolated from mice spleens and activated by plate coated anti-CD3 (10 μg/mL), anti-CD28 (2 μg/mL) antibodies in the presence of TGF-β (10 ng/mL) and IL-2 (2 ng/mL) for 5–7 days. The yield and purity of Treg fraction was monitored by analysis of CD4⁺Foxp3⁺ cells using flow cytometry. iTreg were not restimulated before coculture with the target cells.
Intratumoral Treg cells were isolated from MC38 tumors grown in WT mice using MACS separation kit (Miltenyi Biotec). Isolated Tregs were cocultured with OT-1 cells at the ratio=1:3 and the OT-1 cell toxicity was determined as described in the next section.

Zhang H., Yu P., Tomar V.S., Chen X., Atherton M.J., Lu Z., Zhang H.G., Li S., Ortiz A., Gui J., Leu N.A., Yan F., Blanco A., Meyer-Ficca M.L., Meyer R.G., Beiting D.P., Li J., Nunez-Cruz S., O’Connor R.S., Johnson L.R., Minn A.J., George S.S., Koumenis C., Diehl J.A., Milone M.C., Zheng H, & Fuchs S.Y. (2022). Targeting PARP11 to avert immunosuppression and improve CAR T therapy in solid tumors. Nature cancer, 3(7), 808-820.

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NK Cell Cytotoxicity Assay Using 35S Release

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NK cells were isolated from healthy donors via MACS separation kit (Miltenyibiotech) and grown in the presence of IL-2 (peprotech). Target cells were grown over night in the presence of ³⁵S added to a Methionine-free media (Sigma). Prior to incubation with the effectors, cells were washed, counted, and 5000 cells/well were plated. For each target, the spontaneous ³⁵S release was calculated by cells which were not incubated with effector cells, and maximum ³⁵S release was calculated by applying 0.1M of NaOH to the target cells. The level of ³⁵S release was measured after 5 hours of incubation with effectors (at 37°_c) by a β-counter TopCount (Packard). When blocking antibody was used, NK cells were preincubated with 0.5 ug/well of the blocking antibody on ice for 1 hour, and then the ³⁵S labeled target cells were added for 5-hour incubation.

Nachmani D., Zimmermann A., Oiknine Djian E., Weisblum Y., Livneh Y., Khanh Le V.T., Galun E., Horejsi V., Isakov O., Shomron N., Wolf D.G., Hengel H, & Mandelboim O. (2014). MicroRNA Editing Facilitates Immune Elimination of HCMV Infected Cells. PLoS Pathogens, 10(2), e1003963.

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Isolating and Culturing CD133+ Glioma Spheroids

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10 days after infection, U251 cells were collected and dissociated into a single cell suspension using Stem Pro Accutase (Thermo Fisher). CD133⁺ cells were next isolated using a MACS separation kit (Miltenyi Biotec) following the manufacturer’s protocol and counted under a light microscope. 1000 (for ‘clonal conditions’ or 10000 (for ‘bulk conditions’) CD133⁺ cells were next plated in 6 ml of 3D culture media (DMEMF12 media, 1xP/S, 10 ng/ml EGF, 10 ng/ml FGF, 1x B27 supplement (Gibco)) in duplicates or triplicates and cultured for 4 weeks. 1 ml of fresh 3D culture media was added to the flasks every 10 days. Pictures of the spheroids were taken on the 21^st day of 3D culture. The entire experiment was independently repeated 3 times.

Kuciak M., Mas C., Borges I., Sánchez-Gómez P, & Ruiz i Altaba A. (2019). Chimeric NANOG repressors inhibit glioblastoma growth in vivo in a context-dependent manner. Scientific Reports, 9, 3891.

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Hepatic NK Cell Cytotoxicity Assay

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The hepatic NK cells of infected BALB/cJ mice were isolated using a MACS separation kit (Miltenyi Biotech, Germany) and cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS). The remaining hepatocytes were set as the target. The target-cell only group cultured with 100 μl lysis solution (Fermentas, Glen Burnie, MD) for an hour as the maxium-releasing group and the natural cultured cell group was set as the spontaneous-releasing group. ALT was quantitated using an Automatic Chemistry Analyzer (Hitachi, Tokyo, Japan). The cytotoxicity was defined as ratio of (experimental releasing - spontaneous releasing) / (maxium releasing - spontaneous releasing) * 100%. Triplicate assays were conducted.

Zhang X., Zhu L., Zhou Y., Shi A., Wang H., Han M., Wan X., Kilonzo S.B., Luo X., Chen T, & Ning Q. (2018). Interference with KCTD9 inhibits NK cell activation and ameliorates fulminant liver failure in mice. BMC Immunology, 19, 20.

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In vitro and ex vivo Treg Characterization

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Treg cells were either in vitro differentiated or
isolated from MC38 tumors grown in WT or SA mice using a MACS separation kit
(Miltenyi Biotec, cat#130-091-041) according to the manufacturer’s
instructions. iTreg cells were not re-stimulated before being used either in
cytotoxicity assays or in T-cell proliferation assays. For the cytotoxicity
assay (described above) Treg or iTreg cells were co-cultured with OT-I cells
(1:3 or at indicated conditions) followed by assessment of killing MC38OVA cells
as described above. For T-cell proliferation assays, splenic naïve
CD8⁺ T cells were isolated from WT mice using a kit (Stemcell,
cat#19858), labeled with CellTrace Violet (Thermo fisher, cat#C34557), and
cultured either alone or with the indicated Treg cells (Treg: T = 1:3) in the
presence of magnetic beads precoated with agonist antibodies against CD3 and
CD28 (Gibco, cat#11453D), and proliferation was measured by flow cytometry after
3 days. Proliferation Index is the total number of divisions divided by the
number of cells that went into division, which were analyzed by FlowJo 10.

Zhang H., Tomar V.S., Li J., Basavaraja R., Yan F., Gui J., McBrearty N., Costich T.L., Beiting D.P., Blanco M.A., Conejo-Garcia J.R., Saggu G., Berger A., Nefedova Y., Gabrilovich D.I, & Fuchs S.Y. (2022). Protection of regulatory T cells from fragility and inactivation in the tumor microenvironment. Cancer immunology research, 10(12), 1490-1505.

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