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Streptavidin coated microbeads

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Streptavidin-coated microbeads are small, spherical particles that have streptavidin, a protein with high affinity for biotin, bound to their surface. These microbeads can be used to capture and isolate biotinylated molecules or cells from complex mixtures.

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Lab products found in correlation

Biotinylated anti ly6g antibody, by Miltenyi Biotec (2 mentions) Macs column, by Miltenyi Biotec (2 mentions) Gm csf, by Thermo Fisher Scientific (2 mentions) Anti cd16 32, by BioLegend (2 mentions) Anti mhc class 2, by BioLegend (2 mentions) Anti ter119, by BioLegend (2 mentions) Anti cd19, by BioLegend (2 mentions) Anti cd4, by BioLegend (2 mentions) Macs depletion column, by Miltenyi Biotec (2 mentions) Facsaria 2 cell sorter, by BD (2 mentions) Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (2 mentions) Poly allylamine hydrochloride, by Polysciences (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions) 1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride edc, by Thermo Fisher Scientific (1 mentions) Tween 20, by Merck Group (1 mentions) N hydroxysuccinimide nhs, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Mouse tumor dissociation kit, by Miltenyi Biotec (1 mentions) Moflo astrios eq, by Beckman Coulter (1 mentions) Facsaria 2, by BD (1 mentions)

9 protocols using streptavidin coated microbeads

1

Biofunctionalized Magnetic Beads

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Poly allylamine hydrochloride (Polyscience), poly ethylene-alt-maleic anhydride (Aldrich), phosphate buffered saline (PBS) (Invitrogen), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (Thermo scientific), N-hydroxysuccinimide (NHS) (Aldrich), bovine serum albumin (BSA) (Aldrich), Tween 20 (Aldrich), and streptavidin-coated MicroBeads (Miltenyi, 130-048-101) were used as received, and without further purification.
Xu L., Lee J.R., Hao S., Ling X.B., Brooks J.D., Wang S.X, & Gambhir S.S. (2019). Improved detection of prostate cancer using a magneto-nanosensor assay for serum circulating autoantibodies. PLoS ONE, 14(8), e0221051.
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2

Isolation of Myeloid-Derived Suppressor Cells

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Single-cell suspensions were prepared from the spleen followed by red blood cell removal using ACK buffer. Single-cell suspensions from tumor tissues were prepared using Mouse Tumor Dissociation Kit according to the manufacturer’s recommendation (Miltenyi). For PMN-MDSCs isolation, cells were labeled with biotinylated anti-Ly6G antibody (Miltenyi Biotec), incubated with streptavidin-coated microbeads (Miltenyi Biotec), and separated on MACS columns (Miltenyi Biotec). M-MDSCs were sorted by using either FACS Aria II (BD Biosciences) and MoFlo Astrios EQ (Beckman Coulter) cell sorters. The antibodies for M-MDSCs isolation are described in Supplementary Table 1.
Alicea-Torres K., Sanseviero E., Gui J., Chen J., Veglia F., Yu Q., Donthireddy L., Kossenkov A., Lin C., Fu S., Mulligan C., Nam B., Masters G., Denstman F., Bennett J., Hockstein N., Rynda-Apple A., Nefedova Y., Fuchs S.Y, & Gabrilovich D.I. (2021). Immune suppressive activity of myeloid-derived suppressor cells in cancer requires inactivation of the type I interferon pathway. Nature Communications, 12, 1717.
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3

Isolation and Differentiation of MDSCs from PBMCs

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Human PBMCs were purchased from Cellular Technology Ltd (CTL). For CD33+ PBMC isolation, cells were labeled with biotinylated anti‐CD33 antibody (Biolegend), incubated with streptavidin‐coated microbeads (Miltenyi Biotec) and separated on an MACS columns (Miltenyi Biotec). CD33+ PBMCs were differentiated into MDSCs in the lower chambers of a 24‐well transwell plate (Corning) in complete RPMI‐1640 medium supplemented with IL‐6 (10 ng/mL, Sigma‐Aldrich) and GM‐CSF (10 ng/mL, PeproTech) for 7 days as previously described.23
Kumata S., Notsuda H., Su M., Saito‐Koyama R., Tanaka R., Suzuki Y., Funahashi J., Endo S., Yokota I., Takai T, & Okada Y. (2023). Prognostic impact of LILRB4 expression on tumor‐infiltrating cells in resected non‐small cell lung cancer. Thoracic Cancer, 14(21), 2057-2068.
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4

Differentiation of Human Myeloid Cells from PBMCs

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1.07 × 105 OP9 cells were plated in a 12-well plate the day before sorting. The low-density PBMC fraction was isolated by centrifugation over a Ficoll-Paque Plus density gradient (GE Healthcare Life Sciences). The PBMC layer was collected, and cells were washed and stained with CD14-APC-Cy7, HLA-DR-APC, CD15-PE, and Aqua Live/Dead Fixable 405. After sorting, MEMα without nucleosides was removed from the OP9 cells wells and replaced with IMDM (Thermo Fisher Scientific) supplemented with 20% FBS and 1% penicillin–streptomycin. Up to 3.5 × 105 sorted cells were added per well in a total volume of 3 ml of IMDM in the presence of 100 ng/ml of G-CSF and 25 ng/ml of GM-CSF (Peprotech) or 100 ng/ml of M-CSF. At day 3, 2 ml of fresh media supplemented with fresh cytokines were added on the top. At day 4, cells were harvested by pipetting up and down. Human CD45+ cells were separated from murine OP9 cells by magnetic separation using biotinylated anti-human CD45 antibody (Miltenyi Biotec), streptavidin-coated microbeads (Miltenyi Biotec), and magnetic-activated cell sorting columns (Miltenyi Biotec) according to the manufacturer’s instructions. Cells were then stained with the May-Grünwald-Giemsa kit or the naphthol AS-D chloroacetate esterase (Sigma-Aldrich) kit or stained for flow cytometry.
Mastio J., Condamine T., Dominguez G., Kossenkov A.V., Donthireddy L., Veglia F., Lin C., Wang F., Fu S., Zhou J., Viatour P., Lavilla-Alonso S., Polo A.T., Tcyganov E.N., Mulligan C J.r., Nam B., Bennett J., Masters G., Guarino M., Kumar A., Nefedova Y., Vonderheide R.H., Languino L.R., Abrams S.I, & Gabrilovich D.I. (2019). Identification of monocyte-like precursors of granulocytes in cancer as a mechanism for accumulation of PMN-MDSCs. The Journal of Experimental Medicine, 216(9), 2150-2169.
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5

Adoptive Transfer of gBT-I CD8+ T Cells

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Splenocytes were collected from congenically marked C57Bl6, Thy1.1, or gBT-I CD45.2 mice, and CD8+ T cells were isolated using negative magnetic selection. Splenocytes were incubated with a mixture of biotinylated Abs (anti-CD4, anti-CD19, anti-CD16/32, anti-Ter119, and anti-MHC class II [BioLegend]) and subsequently incubated with streptavidin-coated microbeads (Miltenyi Biotec). Cells were then passed over an LS magnetic column, according to the manufacturer’s instructions. The 1 × 104 CD8-enriched gBT-I splenocytes (85–95% purity) from each male and female adult donor were combined in a 50:50 equal ratio and adoptively transferred (i.v.) into adult B6-Ly5.2 CD45.1 recipient mice the day before infection. At the indicated days postinfection (dpi), the numbers and phenotype of the donor cells in the blood were determined by flow cytometry. The male and female donor CD8+ T cells were distinguished by flow cytometric detection of Thy1.1 and CD45.2. The gating strategy for these in vivo flow cytometric data is CD8+CD4−CD45.2+ CD45.1−KLRG1+CD127− (SLEC) and CD8+CD4−CD45.2+CD45.1− KLRG1−CD127+ (MPEC).
Yee Mon K.J., Goldsmith E., Watson N.B., Wang J., Smith N.L, & Rudd B.D. (2019). Differential Sensitivity to IL-12 Drives Sex-Specific Differences in the CD8+ T Cell Response to Infection. ImmunoHorizons, 3(4), 121-132.
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6

Isolation and Characterization of Hematopoietic and Niche Cells

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To isolate HSPC we used MACS depletion columns (Miltenyi) after incubation with a cocktail of biotin–labeled antibodies (as described in the flow cytometry section) followed by an incubation with streptavidin–coated microbeads (Miltenyi). Then cells were stained with c–kit and sca–1 and LSK were FACS–sorted using a FACSAria II cell sorter (BD Biosystems). To purify niche cells from hematopoietic cells we used MACS depletion columns after incubation with a cocktail of biotin–labeled antibodies as above followed by an incubation with streptavidin–coated microbeads. Then cells were stained with CD45.2, sca–1, CD31 and CD51 (Biolegend, clone RMV–7, 1:100). Endothelial cells were identified as linlow CD45low sca–1high CD31high. Bone marrow MSC were identified as linlow CD45low CD31low sca–1high/intermediate and GFP+. Osteoblasts were linlow CD45low sca–1low CD31low CD51high. For adoptive transfer of GFP+ neutrophils and Ly6Chigh monocytes, bone marrow cells were collected from Ubc–GFP mice for purification of neutrophils and monocytes using MACS depletion columns after incubation with a cocktail of PE–labeled antibodies including B220, CD90, CD49b, NK1.1 and Ter–119 followed by an incubation with PE–coated microbeads. Aortic endothelial cells were identified as CD45.2low CD31high CD107aint/high and FACS–sorted using a FACSAria II cell sorter.
Heidt T., Sager H.B., Courties G., Dutta P., Iwamoto Y., Zaltsman A., von zur Muhlen C., Bode C., Fricchione G.L., Denninger J., Lin C.P., Vinegoni C., Libby P., Swirski F.K., Weissleder R, & Nahrendorf M. (2014). Chronic variable stress activates hematopoietic stem cells. Nature medicine, 20(7), 754-758.
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7

Isolation and Co-culture of Tumor-associated Myeloid Cells

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TAMs, M-MDSCs, and PMN-MDSCs were isolated from tumor tissues by either sorting (TAMs and M-MDSCs) or magnetic separation (PMN-MDSCs). For PMN-MDSC isolation, cells were labeled with biotinylated anti-Ly6G antibody (Miltenyi Biotec), incubated with streptavidin-coated microbeads (Miltenyi Biotec), and separated on magnetic-activated cell sorting columns. Ly6G+ cells obtained after MLPG cultures were isolated in the same way. After isolation, cells were plated in U-bottom 96-well plates in triplicate in complete RMPI without extra cytokines. They were cocultured at different ratios with total splenocytes from Pmel or OT-1 transgenic mice in the presence of cognate peptides: OT-1, SIINFEKL; Pmel, EGSRNQDWL. Cells were incubated for 48 h, and then 3H thymidine (PerkinElmer) was added (1 µl/well) and incubated overnight. Samples were counted with a TopCount NXT instrument (PerkinElmer).
Mastio J., Condamine T., Dominguez G., Kossenkov A.V., Donthireddy L., Veglia F., Lin C., Wang F., Fu S., Zhou J., Viatour P., Lavilla-Alonso S., Polo A.T., Tcyganov E.N., Mulligan C J.r., Nam B., Bennett J., Masters G., Guarino M., Kumar A., Nefedova Y., Vonderheide R.H., Languino L.R., Abrams S.I, & Gabrilovich D.I. (2019). Identification of monocyte-like precursors of granulocytes in cancer as a mechanism for accumulation of PMN-MDSCs. The Journal of Experimental Medicine, 216(9), 2150-2169.
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8

Adoptive Transfer of gBT-I CD8+ T Cells

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Splenocytes were collected from congenically marked C57Bl6, Thy1.1, or gBT-I CD45.2 mice, and CD8+ T cells were isolated using negative magnetic selection. Splenocytes were incubated with a mixture of biotinylated Abs (anti-CD4, anti-CD19, anti-CD16/32, anti-Ter119, and anti-MHC class II [BioLegend]) and subsequently incubated with streptavidin-coated microbeads (Miltenyi Biotec). Cells were then passed over an LS magnetic column, according to the manufacturer’s instructions. The 1 × 104 CD8-enriched gBT-I splenocytes (85–95% purity) from each male and female adult donor were combined in a 50:50 equal ratio and adoptively transferred (i.v.) into adult B6-Ly5.2 CD45.1 recipient mice the day before infection. At the indicated days postinfection (dpi), the numbers and phenotype of the donor cells in the blood were determined by flow cytometry. The male and female donor CD8+ T cells were distinguished by flow cytometric detection of Thy1.1 and CD45.2. The gating strategy for these in vivo flow cytometric data is CD8+CD4−CD45.2+ CD45.1−KLRG1+CD127− (SLEC) and CD8+CD4−CD45.2+CD45.1− KLRG1−CD127+ (MPEC).
Yee Mon K.J., Goldsmith E., Watson N.B., Wang J., Smith N.L, & Rudd B.D. (2019). Differential Sensitivity to IL-12 Drives Sex-Specific Differences in the CD8+ T Cell Response to Infection. ImmunoHorizons, 3(4), 121-132.
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9

Isolation and Characterization of Hematopoietic and Niche Cells

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To isolate HSPC we used MACS depletion columns (Miltenyi) after incubation with a cocktail of biotin–labeled antibodies (as described in the flow cytometry section) followed by an incubation with streptavidin–coated microbeads (Miltenyi). Then cells were stained with c–kit and sca–1 and LSK were FACS–sorted using a FACSAria II cell sorter (BD Biosystems). To purify niche cells from hematopoietic cells we used MACS depletion columns after incubation with a cocktail of biotin–labeled antibodies as above followed by an incubation with streptavidin–coated microbeads. Then cells were stained with CD45.2, sca–1, CD31 and CD51 (Biolegend, clone RMV–7, 1:100). Endothelial cells were identified as linlow CD45low sca–1high CD31high. Bone marrow MSC were identified as linlow CD45low CD31low sca–1high/intermediate and GFP+. Osteoblasts were linlow CD45low sca–1low CD31low CD51high. For adoptive transfer of GFP+ neutrophils and Ly6Chigh monocytes, bone marrow cells were collected from Ubc–GFP mice for purification of neutrophils and monocytes using MACS depletion columns after incubation with a cocktail of PE–labeled antibodies including B220, CD90, CD49b, NK1.1 and Ter–119 followed by an incubation with PE–coated microbeads. Aortic endothelial cells were identified as CD45.2low CD31high CD107aint/high and FACS–sorted using a FACSAria II cell sorter.
Heidt T., Sager H.B., Courties G., Dutta P., Iwamoto Y., Zaltsman A., von zur Muhlen C., Bode C., Fricchione G.L., Denninger J., Lin C.P., Vinegoni C., Libby P., Swirski F.K., Weissleder R, & Nahrendorf M. (2014). Chronic variable stress activates hematopoietic stem cells. Nature medicine, 20(7), 754-758.
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