Streptavidin microbeads

Manufactured by Miltenyi Biotec

Sourced in Germany, United States

Streptavidin microbeads are small, paramagnetic particles coated with the protein streptavidin. Streptavidin has a high affinity for the small molecule biotin, allowing for the efficient separation and isolation of biotin-labeled cells, proteins, or other biomolecules using a magnetic field.

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Negative magnetic streptavidin microbead selection (4 citations) Streptavidin-coated microbeads (9 citations) Streptavidin immunomagnetic microbeads (1 citations) Streptavidin-conjugated microbeads (24 citations) Streptavidin magnetic microbeads (10 citations) MACS streptavidin-microbeads (9 citations) μMACS Streptavidin MicroBeads (7 citations) Streptavidin-conjugated magnetic microbeads (2 citations) MACS streptavidin conjugated microbeads (2 citations)

158 protocols using streptavidin microbeads

Isolation and Analysis of Murine LY6G+ Cells

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LY6G⁺ enriched splenocytes were isolated using a two-step magnetic separation. Briefly, splenocytes were labeled for negative selection with biotinylated antibodies to B220, CD4, CD8, CD11C, DX5, MHC class II, and TER119 (eBioscience) and streptavidin-microbeads (Miltenyi Biotec). After enrichment using the Automacs (Miltenyi Biotec) the negative fraction was labeled with a biotinylated antibody to LY6G (eBioscience) and streptavidin-microbeads (Miltenyi Biotec) and again separated using the Automacs (Miltenyi Biotec). Flow cytometry was used to assess the purity of the LY6G⁺ fraction, and the remaining cells were spun onto slides with a cytospin centrifuge (Harlow Scientific) and stained with Giemsa (Ricca Chemical Company). Cells were then observed with an Olympus confocal microscope at 100x magnification.

Kang H., Corr M., Mansson R., Welinder E., Hedrick S.M, & Stone E.L. (2015). Loss of Murine FOXO3 in Cells of the Myeloid Lineage Enhances Myelopoiesis but Protects from K/BxN-Serum Transfer-Induced Arthritis. PLoS ONE, 10(5), e0126728.

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Purification and Culture of Myeloid Progenitor Cells

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For purification of myeloid progenitor cells, bone marrow cells were collected in DMEM/10% FCS. After red cell lysis, cells were stained with biotin-conjugated antibodies against Ter119, Gr-1, Mac-1, B220, CD4, CD8 and IL-7Rα and incubated with streptavidin-microbeads (Miltenyi Biotech). Lineage positive cells were depleted by magnetic separation using MACS LS columns and QuadroMACS^TM separator magnets (Miltenyi Biotech). Hematopoietic myeloid progenitor cells that were propidium iodide (PI) negative, lineage-negative, Sca-1 negative and c-Kit positive, were sorted and collected by flow cytometry on a FACSAria or Diva (BD Biosciences). For imaging studies, cells were resuspended in StemPro-34 SFM (Invitrogen). Cells were cultured in 10 ng/mL human G-CSF (Neupogen, Amgen), 10 ng/mL recombinant mouse GM-CSF or 10 ng/mL recombinant human GM-CSF.

McArthur K., D’Cruz A.A., Segal D., Lackovic K., Wilks A.F., O’Donnell J.A., Nowell C.J., Gerlic M., Huang D.C., Burns C.J, & Croker B.A. (2017). Defining a therapeutic window for kinase inhibitors in leukemia to avoid neutropenia. Oncotarget, 8(35), 57948-57963.

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Purification of CD4+ T cells and CD11c+ cells

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Spleen cells were purified according to standard protocols as follows. CD4 + T cells were negatively selected using a cocktail of antibody-coated magnetic beads from Miltenyi Biotech (anti-CD8a, anti-CD11b, anti-CD45R, anti-DX5, anti-ter 119), according to manufacturer's instructions, yielding CD4 + cells with >95% purity. CD11c + cells were positively selected with biotin-conjugated anti-CD11c mAb (7D4, PharMingen), streptavidin microbeads (Miltenyi Biotec), followed by 2 consecutive magnetic cell separations using LS columns (Miltenyi Biotec), yielding CD11c + cells with >80% purity.

Zeboudj L., Maître M., Guyonnet L., Laurans L., Joffre J., Lemarie J., Bourcier S., Nour-Eldine W., Guérin C., Friard J., Wakkach A., Fabre E., Tedgui A., Mallat Z., Tharaux P.L, & Ait-Oufella H. (2018). Selective EGF-Receptor Inhibition in CD4⁺ T Cells Induces Anergy and Limits Atherosclerosis. Journal of the American College of Cardiology, 71(2).

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Enrichment and Characterization of SARS-CoV-2 Reactive B Cells

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Splenocytes of TC-mAb mice were pre-treated with anti-FcγRII/III monoclonal antibody and then incubated with biotinylated monoclonal antibodies against mouse CD43, CD90, CD3, c-kit, F4/80, Gr-1, CD4, CD8, CD11b, Ter119, CD93, CD11c, CD138, and human IgD. Class-switched memory B cells were enriched with a MACS system using streptavidin microbeads (Miltenyi Biotec). This was followed by staining with B220-BV786, CD38-AF700, PE-labeled CoV2-S, APC-labeled CoV2-RBD, streptavidin-efluor450, and DAPI. Stained cells were analyzed and sorted as single cells into 96-well plates using a FACSAria instrument (BD Biosciences).

Onodera T., Kita S., Adachi Y., Moriyama S., Sato A., Nomura T., Sakakibara S., Inoue T., Tadokoro T., Anraku Y., Yumoto K., Tian C., Fukuhara H., Sasaki M., Orba Y., Shiwa N., Iwata N., Nagata N., Suzuki T., Sasaki J., Sekizuka T., Tonouchi K., Sun L., Fukushi S., Satofuka H., Kazuki Y., Oshimura M., Kurosaki T., Kuroda M., Matsuura Y., Suzuki T., Sawa H., Hashiguchi T., Maenaka K, & Takahashi Y. (2021). A SARS-CoV-2 antibody broadly neutralizes SARS-related coronaviruses and variants by coordinated recognition of a virus-vulnerable site. Immunity, 54(10), 2385-2398.e10.

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Isolation and Characterization of Hematopoietic Stem Cells

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BM cell suspensions were incubated with the FcBlock reagent (anti-CD16/32, in Rat serum—no. 13551, STEMCELL Technologies), labelled with the biotinylated antibodies anti-CD45R/B220, anti-CD19, anti-CD11b/Mac-1, anti-Ly6G/GR-1, anti-pan-NK, anti-Ly-76/TER and anti-CD3 (full list of antibodies and dyes in Supplementary Table 1), and incubated with Streptavidin microbeads (Miltenyi Biotec), for negative selection of lineage-positive cells by immunomagnetic separation using a MACS column (Miltenyi Biotec). Cells were further incubated in serum-free media (StemSpam SFEM, no. 09650, StemCell Technologies) with 20 μg ml⁻¹ Rhodamine 123 (no. R302, Invitrogen) for 30 min in a water bath at 37 °C (in the dark), cooled, rinsed with HBSS, 5% FBS and then stained with Hoechst 33342 (no. H3570, Invitrogen) at a concentration of 2.5 μg ml⁻¹ diluted in the serum-free media as before, for 90 min at 37 °C, in the dark. Cells were then labelled in HBSS with 5% FBS for 30 min on ice, with Streptavidin-APC and anti-CD45-PE and 1 μg ml⁻¹ Propidium Iodide (no. P3566, Invitrogen). For the long-term reconstituting SP cells gating adjustment, an aliquot of the sample was incubated with 25 mg ml⁻¹ of Verapamil (no. 676777, Calbiochem) before staining with the dyes. Further stainings with different HSC-specific marker combinations were tested, confirming the enrichment of HSCs within the SP gates.

Alves-Pereira C.F., de Freitas R., Lopes T., Gardner R., Marta F., Vieira P, & Barreto V.M. (2014). Independent recruitment of Igh alleles in V(D)J recombination. Nature Communications, 5, 5623.

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Isolation of Murine Monocytes

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Long bones from 6- to 8-week-old C57BL/6 mice were dissected, and the bone marrow was flushed with RPMI 1640 + 2% FCS medium. After red blood cell lysis (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA), monocytes were purified with anti-CD115-biotin antibody (eBioscience, San Diego, CA) coupled to Streptavidin MicroBeads (Miltenyi Biotec). Monocytes (CD115⁺) were used for the transmigration assay.

Roblek M., Strutzmann E., Zankl C., Adage T., Heikenwalder M., Atlic A., Weis R., Kungl A, & Borsig L. (2016). Targeting of CCL2-CCR2-Glycosaminoglycan Axis Using a CCL2 Decoy Protein Attenuates Metastasis through Inhibition of Tumor Cell Seeding. Neoplasia (New York, N.Y.), 18(1), 49-59.

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Suppression of CD8+ T Cell Proliferation by Myeloid Cells

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Ly6G⁺ cells were purified from spleen cells or tumor cells by positive selection using biotinylated Ly6G antibody and streptavidin microbeads (Miltenyi). The purity of the cell populations was >95%. CD11b⁺Ly6C^highLy6G⁻ cells were isolated from spleen cells by cell sorting on a FACSAria cell sorter (BD Biosciences). CD8⁺ T cells from PMEL mice that recognize the gp100-derived peptide, were used as responders. Splenocytes from PMEL mice were mixed with splenocytes from naïve mice at 1:4 ratio in complete RPMI media and plated into 96-well U-bottom plates at 10⁵ cells/well. Ly6G⁺ or CD11b⁺Ly6C^highLy6G⁻ cells were added to the wells at 1:16–1:1 ratios. Murine gp100 peptide (25 (link)–33 (link)) EGSRNQDWL (AnaSpec, Inc.) was added into the wells at the final concentration of 0.1 μg/mL. After 48 hours, cells were pulsed with ³H-thymidine (1 μCi/well; GE healthcare) for 16 hours. ³H-thymidine uptake was counted using a liquid scintillation counter as counts per minute (cpm) and calculated the percentage of proliferation to the positive control (the wells with responder cells and peptide but without suppressive cells).

Hashimoto A., Gao C., Mastio J., Kossenkov A., Abrams S.I., Purandare A.V., Desilva H., Wee S., Hunt J., Jure-Kunkel M, & Gabrilovich D.I. (2018). Inhibition of casein kinase 2 disrupts differentiation of myeloid cells in cancer and enhances the efficacy of immunotherapy in mice. Cancer research, 78(19), 5644-5655.

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In Vitro HSC Lineage Analysis

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For in vitro analyses of HSCs, lineage-depleted bone marrow cells were isolated using streptavidin microbeads (Miltenyi Biotech) and cultured for 4 days in StemSpan medium (StemCell Technologies) supplemented with SCF (10 ng ml⁻¹) and fibroblast growth factor 1 (FGF-1) (10 ng ml⁻¹ all PeproTech). Recombinant mouse CXCL4 (100 ng ml⁻¹; ProsPec) or PBS was added to assess CXCL4 effects on lineage-biased HSCs.

Pinho S., Marchand T., Yang E., Wei Q., Nerlov C, & Frenette P.S. (2018). Lineage-biased hematopoietic stem cells are regulated by distinct niches. Developmental cell, 44(5), 634-641.e4.

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Isolation of Mouse CD4+ T Cell Subsets

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CD4⁺ T cells were negatively selected from mouse spleens and lymph nodes single-cell suspensions after incubation with the following rat anti-mouse hybridoma culture supernatants: anti-MHC class II (M5/114, TIB-120/ATCC; Manassas, VA, United States), anti-CD45R/B220 (RA3-3A1, TIB-146/ATCC), anti-CD16/32 (2.4G2, HB-197/ATCC), anti-CD8 (YTS169; Therapeutic Immunology Group, Oxford, United Kingdom), followed by sheep anti-rat DynaBeads^® (Invitrogen) before separation in a magnetic field. CD4⁺CD25^– and CD4⁺CD25⁺ subsets were selected using anti-CD25-biotinylated (clone 7D4, BD Biosciences) monoclonal antibody (mAb) followed by Streptavidin-MicroBeads^® (MiltenyiBiotec, Germany) and separation on a magnetic column (22 (link)).

Govender L., Mikulic J., Wyss J.C., Gaide O., Thome M, & Golshayan D. (2020). Therapeutic Potential of Targeting Malt1-Dependent TCR Downstream Signaling to Promote the Survival of MHC-Mismatched Allografts. Frontiers in Immunology, 11, 576651.

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Thymus Cell Enrichment and Sorting

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Single-cell suspensions of thymus cells were prepared as described above (flow cytometry) and blocked with anti-CD16/32. The whole thymus cell suspensions were enriched via the depletion of CD4⁺ or CD8⁺ cells by staining with a biotin-conjugated anti-CD4 or CD8α antibody, followed by incubating with streptavidin-microbeads (Miltenyi) and magnetic separation. The enriched cells were stained for surface molecules, and the dead cells were excluded by staining with DAPI. Sorting was performed using an Aria II system (BD Biosciences) and an 85-μm nozzle.

Yoon B.H., Kim M., Kim M.H., Kim H.J., Kim J.H., Kim J.H., Kim J., Kim Y.S., Lee D., Kang S.J, & Kim S.Y. (2018). Dynamic Transcriptome, DNA Methylome, and DNA Hydroxymethylome Networks During T-Cell Lineage Commitment. Molecules and Cells, 41(11), 953-963.

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