Multimacs cell24 separator plus

Manufactured by Miltenyi Biotec

Sourced in Germany

The MultiMACS Cell24 Separator Plus is a laboratory equipment designed for the automated magnetic separation of cells. It provides a versatile and efficient solution for cell isolation and purification processes. The device utilizes magnetic beads to selectively bind and separate target cells from complex mixtures.

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MultiMACS (4 citations)

9 protocols using multimacs cell24 separator plus

Monocyte Isolation and Characterization

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Monocyte isolation was performed using the Pan Monocyte Isolation Kit human (130-096-537, Miltenyi Biotec) and the MultiMACS Cell24 Separator Plus (Miltenyi Biotec) according to the manufacturer’s instructions. Purity of monocyte enrichment was determined by flow-cytometric analysis, using antihuman CD14 (130-110-579, Miltenyi Biotec; 12-0149-42, Invitrogen), antihuman CD16 (130-106-703, Miltenyi Biotec; 17-0168-42, Invitrogen), and the LIVE/DEAD Fixable Aqua Dead Cell Stain kit, for 405-nm excitation (L34966, Invitrogen by Thermo Fisher Scientific). Cells were analyzed using a FACSCanto II flow cytometer and FACSDiva software (BD). Data analysis was performed with FlowJo.

Geisberger S., Bartolomaeus H., Neubert P., Willebrand R., Zasada C., Bartolomaeus T., McParland V., Swinnen D., Geuzens A., Maifeld A., Krampert L., Vogl M., Mähler A., Wilck N., Markó L., Tilic E., Forslund S.K., Binger K.J., Stegbauer J., Dechend R., Kleinewietfeld M., Jantsch J., Kempa S, & Müller D.N. (2021). Salt Transiently Inhibits Mitochondrial Energetics in Mononuclear Phagocytes. Circulation, 144(2), 144-158.

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Saliva and Blood DNA Extraction Protocol

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Blood (whole blood, WB) and saliva samples were collected at the time of the participants' MRI visits. Saliva was self-collected into the Oragene-DNA tubes (DNA Genotek, Ottawa). Salivary DNA was isolated using the prepIT L2P reagent according to the manufacturer's instruction (DNA Genotek, Ottawa), followed by DNA purification and concentration using the Genomic DNA Clean and Concentrator kit (Zymo Research Corporation, Irvine, CA, USA). Venous blood was collected into EDTA anticoagulant lavender top vacutainer tubes and was transferred on ice to the lab as soon as possible. Extraction of genomic DNA from the WB was performed using the Flexi Gene DNA kit according to the manufacturer's protocol (Qiagen, Germany). CD3⁺ cells (T cells) were isolated from WB using CD3 magnetic beads, accompanying reagents, and the MultiMACS Cell24 separator Plus (all from Miltenyi Biotec, Bergisch-Gladbach, Germany). The Allprep DNA/RNA kit (Qiagen, Germany) was then utilized for DNA extraction from CD3⁺ cells according to the manufacturer's instructions.

Burenkova O.V., Naumova O.Y., Church J.A., Juranek J., Fletcher J.M, & Grigorenko E.L. (2023). Associations between telomere length, glucocorticoid receptor gene DNA methylation, volume of stress-related brain structures, and academic performance in middle-school-age children. Comprehensive Psychoneuroendocrinology, 17, 100223.

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Isolation and Purification of Mouse Brain Cells

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Dissociation of cortex and corpus callosum from mice brain was done using neural tissue dissociation kit (P) (Miltenyi Biotec; ref 130-093-231). Briefly, cortices were dissected from P7, P12, P14, or P21 mice and dissociated using a MACS dissociator (Miltenyi Biotec; ref 130-096-427) followed by filtration through a 70 μm cell strainer (Smartstainer; Miltenyi Biotec; ref 130-098-462). Myelin residues were eliminated from P12, P14, and P21 mice cortices during an additional step using the debris removal kit (Miltenyi Biotec; ref 130-090-101). Cells were suspended in a 0.5% NGS solution, then incubated with anti-PDGFRα or anti-O4 coupled-beads (Miltenyi Biotec; ref 130-094-543 and 130-096-670). Unbound bead-coupled antibodies were washed away by centrifugation, leaving bound cells that were sorted using MultiMACS Cell24 Separator Plus (Miltenyi Biotec; ref 130-098-637). Sorted cells were either plated in culture plates for in vitro cell study or centrifuged at 1200 rpm and used for Western blot analysis or ChIP-seq.

Merour E., Hmidan H., Marie C., Helou P.H., Lu H., Potel A., Hure J.B., Clavairoly A., Shih Y.P., Goudarzi S., Dussaud S., Ravassard P., Hafizi S., Lo S.H., Hassan B.A, & Parras C. (2022). Transient regulation of focal adhesion via Tensin3 is required for nascent oligodendrocyte differentiation. eLife, 11, e80273.

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Isolation of Alveolar Epithelial Cells

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Alveolar epithelial cells were sorted using a positive selection of Anti-CD326/EpCam magnetic microbeads (Miltenyi Biotec, 130-105-958, Bergisch Gladbach, Germany). Briefly, lungs were subjected to manual dissection and were perfused with dispase (Corning) and DNase (50 mg/mL; Sigma-Aldrich) to obtain single-cell suspensions. Then, the cell suspension is loaded onto a MACS® Column which is placed in the magnetic field of a separator (Miltenyi Biotec, MultiMACS Cell 24 Separator Plus).

Placido L., Romero Y., Maldonado M., Toscano-Marquez F., Ramírez R., Calyeca J., Mora A.L., Selman M, & Pardo A. (2021). Loss of MT1-MMP in Alveolar Epithelial Cells Exacerbates Pulmonary Fibrosis. International Journal of Molecular Sciences, 22(6), 2923.

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Enrichment of Murine Splenic B Cells

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Spleens were harvested at the indicated time points of the immunization schedule. Spleen cell suspensions were recovered following disassociating using a 40 µm cell strainer. Cellular suspensions were pelleted at 300 g for 5 min, and red blood cell lysis was performed for 1 min using BD Pharm Lyse (BD). Cells were washed twice with MACS buffer and re-suspended in 3 ml of MACS buffer. These cells were further processed according to the manufacturer’s protocol using the Pan B Cell Isolation Kit II (Miltenyi) on a MultiMACS Cell24 Separator Plus (Miltenyi, program depletion). Purity of B-cell lineage was usually above 90% (data not shown).

Molari M., Eyer K., Baudry J., Cocco S, & Monasson R. (2020). Quantitative modeling of the effect of antigen dosage on B-cell affinity distributions in maturating germinal centers. eLife, 9, e55678.

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Profiling Tumor-Infiltrating CD8+ T Cells

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Mice were inoculated with 2.0 × 10⁶ LLC cells on the back and then treated with anti-PD1/CTLA4. On post challenge day 18, tumors were excised, weighed, and chopped before chemical and mechanical digestion with the Tumor Dissociation Kit (Miltenyi) and the gentleMACS Dissociator (Miltenyi) using the m-TDK-2 program. The resulting cell suspension was then passed through a 70 micron filter. Lymphocytes were then enriched using Lympholyte M cell separation media (Cedarlane), per the manufacturer’s instructions. Enrichment for CD45⁺ cells was performed using magnetic beads (Miltenyi) and the MultiMACS Cell24 Separator Plus (Miltenyi). Cells were then stained in PBS using Live/Dead Fixable Near-IR Dead Cell Stain Kit (1:100, ThermoFischer), H-2Kb MuLV p15E Tetramer (1:10, MBL International), and TrueStain FcX Plus (anti-mouse CD16/32) Antibody (1:1000, BioLegend). Samples were then stained for CD45 (1:100, Invitrogen) and CD8 (1:10, MBL International) in MACS Buffer. After washing, cells were fixed and permeabilized using the Fix and Perm Cell Permeabilization Kit (ThermoFischer) before staining for Perforin (1:25, S16009A, Biolegend). Samples were analyzed using a Cytoflex LX instrument (Beckman Coulter) using single-colour compensation controls and fluorescence-minus-one controls for setting gates.

Griffin G.K., Wu J., Iracheta-Vellve A., Patti J.C., Hsu J., Davis T., Dele-Oni D., Du P.P., Halawi A., Ishizuka J.J., Kim S., Klaeger S., Knudsen N.H., Miller B.C., Nguyen T., Olander K., Papanastasiou M., Rachimi S., Robitschek E.J., Schneider E.M., Yeary M., Zimmer M.D., Jaffe J.D., Carr S.A., Doench J.G., Haining W.N., Yates K.B., Manguso R.T, & Bernstein B.E. (2021). Epigenetic Silencing by SETDB1 Suppresses Tumor Intrinsic Immunogenicity. Nature, 595(7866), 309-314.

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Monocyte Transwell Assay for Chemotaxis

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Monocytes were isolated through the positive selection of CD14+ cells directly from anticoagulated whole blood [69 (link)] using the autoMACS Pro Separator, the MultiMACS Cell24 Separator Plus (Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany), or the Whole Blood Column Kit (Whole Blood CD14 MicroBeads human Order no. 130-090-879). Isolated monocytes were seeded on top of a filter insert hanging in a Transwell chamber above PPAs monocytes, and PPAs were co-cultured for 24 h [69 (link)]. Transmigration of monocytes was quantified by the Chemotaxis Cell Migration Assay (QCM Chemotaxis Cell Migration Assay, 24-well, 5 µm, Merck KGaA Darmstadt, Germany). After chemotaxis, migration of monocytes on the lower filter side was measured colorimetrically (560 nm) following staining with crystal violet, a nucleic dye. The mRNA levels of MCP-1 and IL-8 were determined in co-cultured PPAs by real-time PCR, as previously described [2 (link)].

Siegel-Axel D., Barroso Oquendo M., Gerst F., Fend F., Wagner R., Heni M., Königsrainer A., Häring H.U., Fritsche A., Schleicher E., Birkenfeld A.L, & Stefan N. (2023). Extracellular Matrix Expression in Human Pancreatic Fat Cells of Patients with Normal Glucose Regulation, Prediabetes and Type 2 Diabetes. International Journal of Molecular Sciences, 24(13), 11169.

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T Cell Isolation from Leukopaks

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Healthy donor leukopaks were purchased from PPA Research Group (pparesearch.com) and Miltenyi Biotec. Fresh peripheral blood mononuclear cells (PBMCs) were isolated by low-density centrifugation on Lymphoprep (Stem Cell Technology) according to the manufacturer’s instructions. CD3+ T cells were purified by negative selection using the Pan T Cell Isolation Kit Dynabeads Untouched Human T Cells (Invitrogen). In some experiments, T cells were enriched by CD4/CD8 positive selection using the StraightFrom^® Leukopak^® CD4/CD8 MicroBead kit, human (Miltenyi Biotec) and MultiMACS™ Cell24 Separator Plus (Miltenyi Biotec) according to the manufacturer’s instructions.

Li L., Gao Y., Srivastava R., Wang W., Xiong Q., Fang Z., Pelayo A., Denson C., Goswami A., Harari-Steinfeld R., Yang Z., Weng L., Qi L.S, & Marincola F.M. (2020). Lentiviral delivery of combinatorial CAR/CRISPRi circuit into human primary T cells is enhanced by TBK1/IKKɛ complex inhibitor BX795. Journal of Translational Medicine, 18, 363.

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Dissociating Mouse Brain Cortex and Corpus Callosum

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Dissociation of cortex and corpus callosum from mice brain was done using neural tissue dissociation kit (P) (Miltenyi Biotec; . Briefly, cortices were dissected from P7, P12, P14 or P21 mice and dissociated using a MACS dissociator (Miltenyi Biotec; ref 130-096-427) followed by filtration through a 70µm cell strainer (Smartstainer; Miltenyi Biotec; ref 130-098-462). Myelin residues were eliminated from P12, P14 and P21 mice cortices during an additional step using the debris removal kit (Miltenyi Biotec; . Cells were suspended in a 0.5% NGS solution then incubated with anti-PDGFRα or anti-O4 coupled-beads (Miltenyi Biotec; . Unbound bead-coupled antibodies were washed away by centrifugation, leaving bound cells which were sorted using MultiMACS Cell24 Separator Plus (Miltenyi Biotec; . Sorted cells were either plated in culture plates for in vitro cell study or centrifuged at 1200 rpm and used for Western blot analysis or ChIP-seq.

Merour E., Hmidan H., Marie C., Helou P., Lu H., Potel A., Huré J., Clavairoly A., Shih Y., Goudarzi S., Dussaud S., Czernichow P., Hafizi S., Lo S.H., Gelbart W.M., & Parras C. (2022). Transient regulation of focal adhesion via Tensin3 is required for nascent oligodendrocyte differentiation.

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