Automacs magnetic separation system

Manufactured by Miltenyi Biotec

The AutoMacs magnetic separation system is a lab equipment designed for automated magnetic cell separation. It utilizes magnetic beads to isolate specific cell populations from complex samples. The system provides a standardized and reproducible approach to cell separation, enabling efficient and reliable results.

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Lab products found in correlation

Pan t cell isolation kit, by Miltenyi Biotec (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) T cell isolation kit, by Miltenyi Biotec (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Ionomycin, by Thermo Fisher Scientific (1 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions) Ionomycin, by Merck Group (1 mentions) Facsaria 2, by BD (1 mentions) Anti cd4 microbeads, by Miltenyi Biotec (1 mentions) Ficoll gradient, by Harvard Bioscience (1 mentions) Cd45ra, by Beckman Coulter (1 mentions) Buffer el, by Qiagen (1 mentions) Horse serum, by Thermo Fisher Scientific (1 mentions) Nerve growth factor (ngf), by Merck Group (1 mentions) Horse serum, by Merck Group (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) L glutamine, by Merck Group (1 mentions) Anti apc microbeads, by Miltenyi Biotec (1 mentions) Bradford assay, by Carl Roth (1 mentions) Luminol detection system, by Carl Roth (1 mentions)

8 protocols using automacs magnetic separation system

Isolation and Enrichment of Murine CD8+ T Cells

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Cell suspensions of the spleen were obtained by mechanically disrupting the tissue with a syringe plunger in cold RPMI 1640. Red blood cells were removed using ACK buffer. Splenocytes were washed in cell culture medium (RPMI 1640) and filtered through a 70 μm nylon cell strainer. Cell concentrations were determined with an automatic animal cell counter and splenocytes were adjusted to a desired final concentration.
CD8⁺ T cells were enriched from pooled spleen by anti-CD8 (Ly-2) mAbs (Miltenyi Biotec, Auburn, CA) and separated using the AutoMACS magnetic separation system (Miltenyi Biotec, Auburn, CA).

Vasquez M., Paredes-Cervantes V., Aranda F., Ardaiz N., Gomar C, & Berraondo P. (2016). Antitumor effect of an adeno-associated virus expressing apolipoprotein A-1 fused to interferon alpha in an interferon alpha-resistant murine tumor model. Oncotarget, 8(3), 5247-5255.

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SILAC-based Proteomics of Primary T Cells

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Primary CD4 + T cells were isolated from three healthy human donors using a Pan T-cell Isolation Kit and AutoMacs magnetic separation system (Miltenyi Biotec). Approval for these studies was obtained from the Ethics Committee of the Medical Faculty at the Otto-von-Guericke University, Magdeburg, Germany. Informed consent was obtained in accordance with the Declaration of Helsinki. Following isolation, primary T cells were kept overnight at a density of 2 × 10⁶ cells/ml at 37 °C with 5% CO₂, then pelleted at 300 × g, 4 °C, 10 min and frozen at −80 °C until lysis. Jurkat T cells (clone E6-1) were cultured in RPMI 1640 SILAC medium with 10% dialyzed FBS (SILAC Quantification Kit, Pierce) and either light arginine and lysine or heavy-labeled ¹³C₆,¹⁵N₄-arginine and ¹³C₆-lysine (Silantes) for 9 days at 37 °C with an atmosphere of 5% CO₂. Jurkat cells were harvested at a density of 1 × 10⁶ cells/ml by spinning at 300 × g, 4 °C, 10 min, then frozen at −80 °C before lysis.

Morrison E., Kuropka B., Kliche S., Brügger B., Krause E, & Freund C. (2015). Quantitative analysis of the human T cell palmitome. Scientific Reports, 5, 11598.

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Isolation and Characterization of Murine T-Cell Subsets

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CD4⁺ T cells were enriched from spleen and lymph nodes of aged female Balb/c mice by magnetic-activated cell sorting (MACS) using anti-CD4 MicroBeads and the autoMACS magnetic separation system (Miltenyi Biotec). Enriched CD4⁺ T cells were stimulated for 3 h with phorbol 12-myristate 13-acetate (PMA; 10 ng/ml, Sigma-Aldrich) and ionomycin (500 ng/ml, Sigma-Aldrich), followed by the labeling of IL-17A and/or IFN-γ-secreting cells using the IL-17A and IFN-γ secretion assay kit (Miltenyi Biotec) according to the manufacturer's instructions. Subsequently, the cells were stained with fluorochrome-conjugated anti-CD3 (145-2C11), CD4 (RM4-5) and CD45RB (C363.16A) antibodies. Th17 cells (CD3⁺CD4⁺CD45RB^lowIL-17A⁺IFN-γ⁻), Th1 cells (CD3⁺CD4⁺CD45RB^lowIL-17A⁻IFN-γ⁺), Th-mem cells (CD3⁺CD4⁺CD45RB^lowIL-17A⁻IFN-γ⁻) and naive CD4⁺ T cells (CD3⁺CD4⁺CD45RB^highIL-17A⁻IFN-γ⁻) were sorted by fluorescence-activated cell sorting (FACS) on a FACSAria II (BD Biosciences). Purities of sorted populations ranged between 97% and 99%. Tregs were sorted from the same source as CD3⁺CD4⁺CD25^high cells (anti-CD25 antibody PC61.5) and subsequently stimulated with the same concentration of PMA/ionomycin for 3 h. Treg purity was confirmed by intracellular staining for Foxp3 (FJK-16S), IL-17A (eBio17B7) and IFN-γ (XMG1.2) using the Foxp3 transcription factor staining buffer set (eBioscience).

Yang B.H., Floess S., Hagemann S., Deyneko I.V., Groebe L., Pezoldt J., Sparwasser T., Lochner M, & Huehn J. (2015). Development of a unique epigenetic signature during in vivo Th17 differentiation. Nucleic Acids Research, 43(3), 1537-1548.

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Isolation and Activation of Human T Cells

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Peripheral blood was obtained from healthy donors. Approval for the studies with human T cells was obtained from the local ethics committee of the Medical Faculty of the Otto-von-Guericke University Magdeburg with the permission number [107/09]. Blood donors gave written informed consent. Mononuclear cells were isolated by Ficoll gradient (Biochrom) centrifugation of heparinized blood. Human T cells were purified by negative selection with the Pan T-cell Isolation Kit according to manufactures instructions and AutoMacs magnetic separation system (Miltenyi Biotec). The purity of T cells was analyzed by flow cytometry and was usually more than 96%. T cells were activated with CD3 antibody (clone OKT3). Plate-bound antibodies were provided as follows.

Mirdamadi Y., Bommhardt U., Goihl A., Guttek K., Zouboulis C.C., Quist S, & Gollnick H. (2017). Insulin and Insulin-like growth factor-1 can activate the phosphoinositide-3-kinase /Akt/FoxO1 pathway in T cells in vitro. Dermato-endocrinology, 9(1), e1356518.

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Isolation of CD4+ Central Memory T Cells

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Buffy coats from female donors were obtained from the DRK Blutspendedienst, Berlin, Germany. Use of buffy coats for scientific purposes in the DEEP project was approved by the ethics committee of the Charite Universitaetsmedizin Berlin (Ethikkommission, Ethikausschuss 1 am Campus Charite Mitte) under the application number EA1/095/13. Informed consent was given by the donors. Peripheral blood mononuclear cells were separated using a LSM 1077 density gradient (PAA, GE Healthcare). Remaining erythrocytes were lysed using the Buffer EL (Qiagen). CD4⁺ T cells were pre-enriched using anti-CD4 microbeads and the AutoMACS magnetic separation system (Miltenyi Biotec). MACS-sorted CD4⁺ T cells were surface stained using the following directly conjugated antibodies (all purchased from BioLegend, except CD45RA that was purchased from Beckman Coulter): CD3 (clone: UCHT1), CD4 (OKT4), CD25 (BC96), CD45RA (2H4LDH11LDB9), CD62L (DREG-56), CD127 (A019D5). The CD4⁺ central memory T cells were sorted as outlined in Supplementary Fig. 16a according to the surface marker combination CD3⁺CD4⁺CD25^lowCD45RA⁻CD62L⁺ by FACS (Aria, BD-Bioscience). Purity of the sorted populations was confirmed by flow cytometry (Supplementary Fig. 16a). TCMs from three donors were pooled and then used to perform the bivalency ChIP, RNA-seq and whole-genome bisulfite sequencing experiments.

Kinkley S., Helmuth J., Polansky J.K., Dunkel I., Gasparoni G., Fröhler S., Chen W., Walter J., Hamann A, & Chung H.R. (2016). reChIP-seq reveals widespread bivalency of H3K4me3 and H3K27me3 in CD4+ memory T cells. Nature Communications, 7, 12514.

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Transfection and Differentiation Protocols

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HEK-293T cells (supplied by Deutsche Stammsammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ Braunschweig, Germany) were used for testing plasmid constructs. Transfection was done with Lipofectamine^® 2000 (Thermo Scientific) according to the manufacturer’s protocol. For Western Blotting, cells were lyzed 48 h after transfection. PC-12 cells were cultured in RPMI medium containing 10% horse serum (v/v), 5% fetal bovine serum (v/v) and 1% L-Glutamine (v/v; all Thermo Scientific). Differentiation was induced with NGF (50 ng/μl; Sigma-Aldrich) under reduced serum condition [RPMI medium containing 0, 2% horse serum (v/v) and 1% L-Glutamine (v/v)]. Splenic CD3⁺ T-cells from mice were purified using T-cell isolation kit and AutoMacs magnetic separation system (Miltenyi Biotec).

Thiere M., Kliche S., Müller B., Teuber J., Nold I, & Stork O. (2016). Integrin Activation Through the Hematopoietic Adapter Molecule ADAP Regulates Dendritic Development of Hippocampal Neurons. Frontiers in Molecular Neuroscience, 9, 91.

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Immunophenotyping of Murine Immune Cells

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CD11b⁺ or CD11b⁻ cells were separated from spleen and lymph nodes of aged female Balb/c mice using APC-conjugated anti-CD11b (M1/70), anti-APC MicroBeads and the autoMACS magnetic separation system (Miltenyi Biotec). MACS-separated CD11b⁻ cells were labeled with antibodies against CD3 (145–2C11), CD4 (RM4–5), CD8 (53–6.7), B220 (RA3–6B2), γδTCR (eBioGL3) and sorted into B cells (CD3⁻B220⁺), CD8⁺ T cells (CD3⁺B220⁻CD4⁻CD8⁺), CD4⁺ T cells (CD3⁺CD4⁺CD8⁻) and γδ T cells (CD3⁺CD4⁻CD8⁻γδTCR⁺). MACS-separated CD11b⁺ cells were stained with antibodies against CD11b, CD11c (N418), CD49b (DX5), Ly6C (HK1.4), Ly6G (1A8), F4/80 (BM8), CD3, CD19 (MB19–1) and sorted into conventional dendritic cells (cDCs) (CD3⁻CD19⁻CD11b^dimCD11c⁺), neutrophils (CD3⁻CD19⁻CD11b⁺CD11c⁻Ly6G⁺), monocytes (CD3⁻CD19⁻CD11b⁺CD11c⁻Ly6G⁻Ly6C⁺), macrophages (CD3⁻CD19⁻CD11b⁺CD11c⁻Ly6G⁻Ly6C⁻ F4/80⁺) and natural killer (NK) cells (CD3⁻CD19⁻CD11b⁺CD11c⁻Ly6G⁻Ly6C⁻ F4/80⁻CD49b⁺).

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Adoptive CD8+ T Cell Transfer Assay

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Splenic CD3 + , CD8 + and CD4 + lymphocytes were purified using T cell isolation kits and autoMACS magnetic separation system (Miltenyi Biotec). Cell lysis was performed as described previously [10, 28] . Equivalent amounts of protein (determined by Bradford assay (50 mg total protein; Carl Roth) were separated by SDS-PAGE and transferred to nitrocellulose. Western blots were conducted with the indicated antibodies and developed with the appropriate HRP-conjugated secondary antibodies (Dianova, Hamburg, Germany) and the Luminol detection system (Carl Roth). The following antibodies and sera were used: the anti-murine ADAP sheep serum (kindly provided by Gary A. Koretzky, University of Pennsylvania, Philadelphia, PA, USA) [29] ; anti-ADAP mAb; anti-SKAP55 mAb (both from BD Biosciences); anti-SKAP-HOM rabbit serum [28] ; and anti-b-actin mAb (Sigma-Aldrich, St. Louis, MO, USA).
Adoptive CD8 + T cell transfer CD8 + T cells were isolated by MACS using the CD8a + T Cell Isolation Kit (Miltenyi Biotec), according to the protocol provided by the manufacturer. The isolated cells were stained with CFSE (final concentration 1.5 mM) for proliferation analysis later on. The purity of the cells was determined by flow cytometry and was .90%. Cells (4 3 10 6 ) were adoptively transferred to WT C57BL/6J recipient mice by intravenous injection into to the tail vein.

Parzmair G.P., Gereke M., Haberkorn O., Annemann M., Podlasly L., Kliche S., Reinhold A., Schraven B, & Bruder D. (2017). ADAP plays a pivotal role in CD4+ T cell activation but is only marginally involved in CD8+ T cell activation, differentiation, and immunity to pathogens. Journal of leukocyte biology, 101(2).

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