Streptavidin magnetic microbeads

Manufactured by Miltenyi Biotec

Sourced in Germany

Streptavidin magnetic microbeads are a type of lab equipment used for various applications in biotechnology and life sciences. Streptavidin, a protein derived from the bacterium Streptomyces, is coupled to magnetic beads, enabling the isolation and separation of biotinylated molecules, cells, or other targets through magnetic separation techniques.

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Streptavidin microbeads (158 citations) Magnetic microbeads (147 citations) Negative magnetic streptavidin microbead selection (4 citations) Streptavidin-conjugated magnetic microbeads (2 citations)

10 protocols using streptavidin magnetic microbeads

Adoptive Transfer of miR-150 CD8+ T Cells

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Single cell suspensions of splenocytes from gBT-I miR-150^−/− and wild-type gBT-I (WT) mice were obtained by manual tissue dissociation through sieves. Untouched, naïve CD8+ cells were isolated by negative magnetic selection: Splenocytes were labeled with biotinylated antibodies to CD4 (GK1.5), CD19 (eBio1D3), CD16/32 (93), MHC-II I-A/I-D (M5/114.15.2) and Ter119 (Ter119) (Ebioscienes or Biolegend). Subsequently, cells bearing these markers were removed using magnetic streptavidin microbeads (Miltenyi). Equal numbers of each type of cell (1 × 10⁴) were co-transferred intravenously (i.v.) into congenic B6-Ly5.2/Cr recipients. The next day we infected mice with either Listeria monocytogenes (Lm-gB) (5 × 10³ CFU, i.v.) or vaccinia virus (VACV-gB) (2 × 10⁵ PFU, i.p.), as previously described29 (link)30 (link). For co-transfers of phenotypically equivalent populations of miR-150^−/− and WT CD8+ T cells, cells were magnetically enriched for untouched CD8+ cells and then sorted with an Aria cell sorter based on CD44 and CD122 expression patterns. Then equal numbers of miR-150^−/− and WT cells from each corresponding subset were transferred into recipient mice, which were subsequently infected with LM-gB.

Smith N.L., Wissink E.M., Grimson A, & Rudd B.D. (2015). miR-150 Regulates Differentiation and Cytolytic Effector Function in CD8+ T cells. Scientific Reports, 5, 16399.

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Comprehensive Immune Cell Profiling

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Blood was collected into EDTA-containing tubes via retro-orbital injection (under anesthesia) or cardiac puncture (from euthanized animals). Red blood cells were lysed using ACK lysis buffer (Thermo Fisher), and whole blood was stained with fluorochrome-conjugated antibodies. Tumor cells were isolated, dissociated in DMEM/F12 media containing collagenase 20mg/ml (Worthington) for 60 mins at 37°C. Digested materials were passed through 100- and 70-µm nylon cell strainers and single-cell suspensions were prepared for subsequent staining. 20µl of Accucheck counting beads (Thermo Fisher) were added to calculate absolute cell numbers. For T cell analysis, single-cell suspensions were incubated with a biotinylated anti-TCRβ antibody for 20 mins at 4°C in the dark, followed by incubation with magnetic Streptavidin Microbeads (Miltenyi Biotec) for 15 mins at 4°C in the dark. Cells were passed through LS column per the manufacturer’s instructions. Isolated TCRβ⁺cells were blocked from non-specific staining with anti-mouse CD16/32 and then stained with fixable viability dye and the indicated antibodies. Antibodies are listed in Supplementary Table 1. Data were acquired on BD Symphony flow cytometer. Compensation, analysis, and visualization of the flow cytometry data were performed using FlowJo^™ v10.8 Software (BD Life Sciences, RRID:SCR_008520).

Kim D., An L., Moon J., Maymi V.I., McGurk A.I., Rudd B.D., Fowell D.J, & White A.C. (2023). Ccr2+ monocyte-derived macrophages influence trajectories of acquired therapy resistance in Braf-mutant melanoma. Cancer research, 83(14), 2328-2344.

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Isolation and Characterization of Murine Myeloid Progenitors

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Bone marrow cells were extracted from the femurs and tibias of C57Bl/6 mice. Bone marrow cells were washed three times with DPBS supplemented with 0.5 mM EDTA and 0.5% BSA. Bone marrow cells were stained using 1:100 dilutions of biotinylated antibodies recognizing CD11b, GR1, B220, and TER119 antibodies (BD Pharmingen, Mississauga, ON). The cells were then labelled with magnetic streptavidin microbeads according to the manufacturer's instructions (Miltenyi Biotec, Auburn, CA). After washing, cells were lineage depleted using. LD columns (Miltenyi Biotec, Auburn, CA). Unfractionated and lineage-depleted (Lin -) cells were labelled with phycoerythrin (PE) conjugated antibodies for CD11b and c-Kit (BD Biosciences, Mississauga, ON), and analyzed by flow cytometry. Lin -bone marrow cells were grown in culture with complete IMDM medium supplemented with 10 ng/ml M-CSF (Peprotech, QC).

Rhee J., Solomon L.A, & DeKoter R.P. (2019). A role for ATP Citrate Lyase in cell cycle regulation during myeloid differentiation. Blood cells, molecules & diseases, 76.

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Isolation and Expansion of Murine Synovial Fibroblasts

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Isolation and ex vivo expansion of murine SFs was done as previously described (27 ). Briefly, paws were harvested from mice, skin and soft tissue were removed, synovial tissue was dissected and digested with type II collagenase (1 mg/ml; Sigma #C6885) for 80 minutes at 37°C. Samples were vortexed vigorously to release cells and centrifuged. For ex vivo expansion, cells were resuspended in DMEM (#21969-035) supplemented with 10% fetal calf serum (FCS; #10270106), 1% penicillin and streptavidin (#15140122), 1% L-glutamine (#25030-024) and 1% NEAA (#11140-035, all from Invitrogen, UK) and cultured in 5% CO2 at 37°C for 24 hours, when culture medium was replaced. Media was changed twice a week and the cells were passaged at 90% confluence using trypsin-EDTA (Invitrogen, #25300-054). Prior to experimental setup, expression of Podoplanin (PDPN, Biolegend, #156204) and CD11b (Invitrogen, #11-0112-85) was assessed by flow cytometry. Myeloid CD11b+ cells were labelled with biotinylated anti-CD11b antibody (Biolegend, #101204) and subsequently depleted using Streptavidin magnetic MicroBeads (MACS Miltenyi Biotec, #130-090-485). For in vitro cytokine stimulation, recombinant IL-1β was used at the indicated concentrations for 6 hours in cDMEM.

Wang Y., Pan P., Khan A., Çil Ç, & Pineda M.A. (2022). Synovial Fibroblast Sialylation Regulates Cell Migration and Activation of Inflammatory Pathways in Arthritogenesis. Frontiers in Immunology, 13, 847581.

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B Cell Activation and IgG1 Quantification

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B cells were isolated from spleens of C57BL/6 mice and enriched through a negative selection in AUTOMACs with biotinylated anti-CD43 antibody (BD Pharmigen, Cat # 5532269) and streptavidin magnetic microbeads (Miltenyi Biotech, Cat # 130-048-102). Purified B cells were transduced for 24 hr in X-vivo medium (Lonza) containing 2 mM L-glutamine, 50 μM ß-mercaptoethanol, 5 ng/ml IL-4 (R&D Systems, cat# 404-ML-010) and 1 μg/mL anti-CD40 antibody (BioLegend, Cat# 102802) in 100 μl total volume in a round bottom 96-well plate, then transferred at 24 hr to supplemented RPMI (see above) containing 5 ng/ml IL-4 and 1 μg/ml anti-CD40 antibody. Cells were cultured for 4–5 days, stained with anti-IgG1 (FITC anti-mouse IgG1; BioLegend, Cat# 406605), and surface IgG1 quantified by flow-cytometry.

Le Q, & Maizels N. (2015). Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID. PLoS Genetics, 11(9), e1005411.

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Isolation and Purification of PMN-MDSCs and M-MDSCs

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PMN-MDSCs were enriched from splenocytes and BM using a magnetic column separation system (Miltenyi Biotec). Cells were pooled from 6 to 8 mice per group. Briefly, cells were first labeled with anti-Ly6G conjugated to biotin (30 minutes at 4°C), then washed and labeled (45 minutes at 4°C) with streptavidin magnetic microbeads (Miltenyi Biotec 130-048-101), washed and loaded onto a column placed in the magnetic field. The positive (PMN-MDSC) and negative cell populations were isolated according to the manufacturer's directions (Miltenyi Biotec). M-MDSCs were enriched from the negative cell population using the EasySep Monocyte Isolation kit (StemCell Technologies, 19861) according to the manufacturer's instructions. Then, the cells were stained for Ly6G, Ly6C, CD11b, and CD45.2 in PBS containing 1.5% FCS and 2 mmol/L EDTA. Ly6G−Ly6ChiCD11b+ (M-MDSCs) and Ly6G+Ly6ClowCD11b+ (PMN-MDSCs) were sorted using FACS ARIA III (BD). Single-cell suspensions from colons were prepared as described above and directly stained for sorting. Samples were counted using a TC20 automated cell counter (Bio-Rad). All cell populations were above 90% purity.

Ashkenazi-Preiser H., Reuven O., Uzan-Yulzari A., Komisarov S., Cirkin R., Turjeman S., Even C., Twaik N., Ben-Meir K., Mikula I J.r., Cohen-Daniel L., Meirow Y., Pikarsky E., Louzoun Y., Koren O, & Baniyash M. (2024). The Cross-talk Between Intestinal Microbiota and MDSCs Fuels Colitis-associated Cancer Development. Cancer Research Communications, 4(4), 1063-1081.

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Isolation of Murine Alveolar Type II Cells

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AT-II cells were purified as described previously (51 (link)). Briefly, after induction of VILI, mice were euthanized by pentobarbital overdose. Lungs were lavaged with 3 × 1 ml of sterile PBS and then perfused with ice-cold saline via the right ventricle. As previously described, 3 ml of prewarmed dispase (BD) was instilled, followed by 0.5 ml of a low-melting point agarose, and chest cavity was iced for 2 min to harden the agarose plug. Lungs were removed intact and incubated in 1 ml of dispase at 37°C for 15 min. Tissue was dissociated manually, and cells were filtered (70/40 μm; Thermo Fisher), followed by centrifugation. Pelleted cells were resuspended and labeled with CD45 MicroBeads (Miltenyi Biotec), followed by negative selection using magnetic sorting technology. CD45-negative cells were next treated with Fc receptor-blocking monoclonal antibody (eBioscience) and labeled with anti-epithelial cell adhesion molecule (EpCAM) biotin (eBioscience) and anti-biotin DyLight 633. EpCAM-positive cells were positively selected using streptavidin magnetic microbeads (Miltenyi). Successful purification of AT-II cells was confirmed by analyzing cells for EpCAM expression using flow cytometric analysis (fig. S4C).

Neudecker V., Brodsky K.S., Clambey E.T., Schmidt E.P., Packard T.A., Davenport B., Standiford T.J., Weng T., Fletcher A.A., Barthel L., Masterson J.C., Furuta G.T., Cai C., Blackburn M.R., Ginde A.A., Graner M.W., Janssen W.J., Zemans R.L., Evans C.M., Burnham E.L., Homann D., Moss M., Kreth S., Zacharowski K., Henson P.M, & Eltzschig H.K. (2017). Neutrophil transfer of miR-223 to lung epithelial cells dampens acute lung injury in mice. Science translational medicine, 9(408), eaah5360.

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Quantification of Donor Dendritic Cells

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For quantification of donor DC numbers, graft livers were digested with Liberase™ (Roche Diagnostics, Indianapolis, IN, USA), and cells were isolated, and then purified by a density gradient separation using OptiPrep (AXIS-SHIELD, Oslo, Norway) as described (3 (link)). Low-density cell suspensions were preincubated with FcγIIR-blocking mAb (clone D34-485; BD Biosciences, San Jose, CA, USA). After incubation with biotin-conjugated anti-donor MHCI mAb and streptavidin magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), cells were positively isolated by autoMACS (Miltenyi Biotec). The isolated cells were stained for Alexa-Fluor 647-conjugated anti-donor MHCII mAb and FITC-, R-phycoerythrin- and PerCP/Cy5.5-conjugated mAbs. Cells were washed twice and acquired on a FACS Calibur (BD Biosciences). Data were analyzed using FlowJo ver.9.2 software (FlowJo LLC, Ashland, OR, USA).

Ueta H., Xu X.D., Yu B., Kitazawa Y., Yu E., Hara Y., Morita-Nakagawa M., Zhou S., Sawanobori Y., Ueha S., Rokutan K., Tanaka T., Tokuda N., Matsushima K, & Matsuno K. (2020). Suppression of liver transplant rejection by anti-donor MHC antibodies via depletion of donor immunogenic dendritic cells. International Immunology, 33(5), 261-272.

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HSC Transplantation and Tracking

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Tibias, femurs, ileac bones, vertebrae and sternum were harvested from donor mice, crushed and filtered through a 40 μm strainer. Whole BM cells were labeled with a cocktail of biotinilated lineage antibodies (CD3, CD4, CD8, Ter119, B220, Ly6G, CD11b) and streptavidin magnetic Microbeads (Miltenyi Biotech) to perform a lineage depletion using the MACS^® Column Technology (Miltenyi Biotech). The lineage-depleted sample was stained and sorted for phenotypic HSCs, defined as lineage^-cKit⁺Sca-1⁺CD48^-CD150⁺ cells. HSCs were centrifuged, suspended in PBS and incubated with Vybrant^® 1,1’-dioctadecyl-3,3,3′-tetramethylindodicarbocyanine perchlorate (DiD) (ThermoFisher Scientific) for 10 min, at 37°C. After washing, 5,000 to 12,000 DiD-labeled HSCs were transplanted per mouse via tail vein injection. Control and leukemic recipient mice had been previously lethally irradiated (two irradiations of 5.5Gy, 3 hours apart). 2 days after transplantation, recipient mice were sacrificed and femurs, tibias and the spleen harvested. DiD⁺DAPI^-cKit⁺Sca-1⁺CD48^-CD150⁺ cells were detected by flow cytometry.

Duarte D., Hawkins E.D., Akinduro O., Ang H., De Filippo K., Kong I.Y., Haltalli M., Ruivo N., Straszkowski L., Vervoort S.J., McLean C., Weber T.S., Khorshed R., Pirillo C., Wei A., Ramasamy S.K., Kusumbe A.P., Duffy K., Adams R.H., Purton L.E., Carlin L.M, & Lo Celso C. (2018). Inhibition of Endosteal Vascular Niche Remodeling Rescues Hematopoietic Stem Cell Loss in AML. Cell Stem Cell, 22(1), 64-77.e6.

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Adoptive Transfer of Ly6C+ Monocytes

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Splenocytes from CD45.2⁺ CCR2^gfp/+ mice were filtered through a 70-µm strainer, and red blood cells were removed by lysis buffer (BD). Taking advantage of biotinylated anti-CD115 antibody (eBioscience) and streptavidin-magnetic microbeads (Miltenyi Biotech), splenic monocytes were enriched using a MACS LS column. Cells were stained with anti-CD11b, anti-CD115, and anti-Ly6C antibodies, and Ly6C^hi monocytes expressing GFP were sorted using a FACSAria II system (BD). Sorted cells (>95% purity) were injected i.v. into congenic CD45.1 mice (1.2 × 10⁶ cells/mouse). Recipient mice were sacrificed and analyzed at day 2.5 or day 4 after injection.

Kim K.W., Williams J.W., Wang Y.T., Ivanov S., Gilfillan S., Colonna M., Virgin H.W., Gautier E.L, & Randolph G.J. (2016). MHC II+ resident peritoneal and pleural macrophages rely on IRF4 for development from circulating monocytes. The Journal of Experimental Medicine, 213(10), 1951-1959.

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