Frozen BM aspirates were thawed on ice and stained with biotinylated antibodies against CD45 (1:50, clone HI30; BioLegend) and CD235a (1:50, clone HIR2, BioLegend) followed by depletion using magnetic anti-biotin beads (20 μL per 107 cells; Miltenyi Biotec) in PBS supplemented with 2% FCS and iMag (BD Biosciences). After depletion, the remaining cells were stained in PBS containing 0.5% FCS at 4°C with the following antibodies: Streptavidin-AF488 (1:100, Invitrogen), CD45-BV510(1:50, clone HI30; BioLegend), CD235a-PE-Cy7(1:50, clone HI264; BioLegend), CD71-AF700 (1:20, clone MEM-75; Exbio), CD271-PE (1:50, clone ME20.4; BioLegend), CD31-APC-Cy7 (1:20, clone WM59; BioLegend), and CD44-APC (1:50, clone IM7, Sony). 7AAD (1:100; Beckman Coulter) was used for dead cell exclusion. Samples were measured using FACSymphony A5 Cell Analyzer and analyzed with a FlowJo_v10.6.1 program.
Anti biotin magnetic beads
Manufactured by Miltenyi Biotec
Sourced in United States, Germany
Anti-biotin magnetic beads are a type of lab equipment used for the separation and purification of biotinylated molecules. They consist of magnetic particles coated with anti-biotin antibodies, which can efficiently bind and capture biotin-labeled targets. These beads provide a convenient and effective method for isolating and concentrating specific biomolecules from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Automacs separator, by Miltenyi Biotec (4 mentions)
Facsaria 2, by BD (3 mentions)
Macs ls column, by Miltenyi Biotec (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Anti cd11b, by BD (2 mentions)
Ls column, by Miltenyi Biotec (2 mentions)
Cd45 bv510, by BioLegend (1 mentions)
Streptavidin af488, by Thermo Fisher Scientific (1 mentions)
Clone hi30, by BioLegend (1 mentions)
Cd31 apc cy7, by BioLegend (1 mentions)
Cd235a pe cy7, by BioLegend (1 mentions)
Clone hir2, by BioLegend (1 mentions)
7 aad, by Beckman Coulter (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Brefeldin a, by BD (1 mentions)
Macs ld column, by Miltenyi Biotec (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Anti cd8α magnetic beads, by Miltenyi Biotec (1 mentions)
38 protocols using anti biotin magnetic beads
1
Immunophenotyping of Hematopoietic Subsets
2
Purification and Activation of OT II T Cells
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+ T cells were purified from spleens of OT II mice by MACS (Miltenyi Biotec) according to the manufacturer's protocol. Briefly, cells were Fc-blocked and incubated with biotinylated anti-CD4 antibodies (BD Pharmingen). Subsequently, magnetic anti-biotin beads (Miltenyi Biotec) were added and CD4 + T cells were positively selected by running cells along a MACS magnet. CD4 + T cells were labeled with CellTrace Violet (Thermo Fisher Scientific) and afterwards cultured with untreated or BEA treated BMDCs at a 10:1 ratio in 96 well round bottom plates for 3 days and 5 days, respectively. Cell proliferation was measured at day 3 by flow cytometry. At day 5, cell culture supernatants were collected and stored at -80°C. For intracellular detection of IFNγ, Brefeldin A (BD Biosciences) was added to the cells in the last 6 hours before harvesting of the cells at day 5.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
3
Isolation of Inflammatory and Bone Marrow Neutrophils
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Peritoneal neutrophils (herein referred as inflammatory neutrophils) were obtained by i.p. injection of 10% thioglycollate (SIGMA), as described [28 (link)]. Cells were collected 18 h later, by peritoneal washings, counted, and were left to adhere for 1h at 37°C. Non-adherent cells were recovered, washed and examined for purity by both FACS and H&E staining of cytospin preparations. Neutrophils of purity >90% were used in experiments. Bone marrow neutrophils were obtained from the tibia and femur of mice; labeled with neutrophil-specific mAbs anti-Ly6G (clone NIMP-R14-FITC or clone-1A8-PE) (BD PharMingen) and purified by MACS-positive selection, using using anti-FITC or anti-PE magnetic beads (Miltenyi Biotech). Alternatively, neutrophils were labeled with anti-Ly6G (clone 1A8, conjugated to Biotin, Miltenyi Biotech) and purified using anti-Biotin magnetic beads. Purity of neutrophils following either NIMP-R14 or 1A8 positive MACS selection was > 95%, as assessed by FACS. Control stainings with CD11b and Ly6C were performed following magnetic separation and neutrophils (inflammatory and bone marrow) were characterized as CD11b+1A8+Ly6Cint and Gr1high (S1 and S2 Figs, respectively).
4
Isolation of Immune Cell Subsets
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Murine spleen, thymus, lymph node, bone marrow, and lung were homogenized using a cell strainer, and red blood cells (RBC) were lysed using an RBC lysis buffer. Hepatic lymphocytes were isolated using Percoll (GE Healthcare, Little Chalfont, UK) as previously described (Lee et al., 2012 ). Peripheral blood mononuclear cells (PBMC) were isolated using Histopaque‐1077 (Sigma‐Aldrich, St. Louis, MO, USA) following the manufacturer's protocol. In several in vitro and in vivo experiments, splenic CD8+ T cells were enriched using anti‐CD8α+ magnetic beads and a MACS LS column (Miltenyi Biotec, Bergisch Gladbach, Germany), then sorted into three subsets, Tim‐3+PD‐1+, Tim‐3−PD‐1+, and Tim‐3−PD‐1− by FACSAria II (BD Biosciences, San Jose, CA, USA). The sort purities were more than 95%. To prepare the T‐cell‐depleted antigen presenting cells (APCs), splenocytes were stained with biotinylated anti‐CD3 mAb (Biolegend, San Diego, CA, USA) and antibiotin magnetic beads (Miltenyi Biotec), after which an MACS LD column (Miltenyi Biotec) was used.
5
Bone Marrow Progenitor Cell Isolation
6
Isolation of Naive CD4+ T Cells
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Single-cell leukocyte suspensions were obtained from spleens of 5- to 10-week-old 5C.C7 Rag2−/−, T-bet−/− or WT mice. Depletion of non-CD4+ T cells was done using biotin-conjugated monoclonal anti-mouse antibodies against CD8α, CD11b, CD45R, CD49b, Ter-119 and anti-biotin magnetic beads (Miltenyi Biotec). Cells were further sorted using α-CD4-PE and α-PE magnetic beads (Miltenyi Biotec) and CD4+CD25-CD44lowCD62Lhigh (all eBioscience) cells were sorted by flow cytometry. All antibodies were used at a concentration of 1–2 μg per 1 × 106 cells. Purities of CD4+CD25-CD44lowCD62Lhigh T cells after isolation were >99.2%.
7
Magnetic Cell Isolation for Metabolic Analysis
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To isolate specific populations of cells, single-cell suspensions as isolated above are preblocked with anti-CD16/32 for 15 min at 4°C. We then used the biotinylated anti-Gr1 (clone RB6-8C5), anti-CD8β, or anti-CD163 antibodies (all from Thermo Fisher Scientific) to label murine myeloid, CD8+, and human monocytes, respectively. Next, the cells were washed and then incubated with anti-biotin magnetic beads (Miltenyi Biotec) before performing manual positive selection using MS columns (Miltenyi Biotec). Purified cells were analyzed for all downstream metabolic analyses.
8
Isolation and Characterization of Effector T Cells
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Fresh total cells from spleen were isolated in PBS1X-3% fetal calf serum and stained for 20 min at 4°C with anti-Ter-119-biotin, anti-CD11c-biotin, and anti-B220-biotin antibodies followed by labeling with anti-biotin magnetic beads (Miltenyi Biotec) for 15 min at 4°C. B cells and erythrocytes were depleted on an AutoMACS separator (Miltenyi Biotec) following the manufacturer’s procedure. Enriched T cells were stained with anti-CD3 APC, anti-CD4 Horizon V500, anti-CD8 Alexa 700, anti-CD44 PE, and anti-CD62L efluor 450. 6.105 CD3+CD4+GFP− Teff cells were sorted on a BD FACSAria II (BD Biosciences, San Jose, CA, USA) with a purity >99%. Sorted cells were stored in Trizol (Invitrogen) or RNAAquous (Ambion, Inc./Life Technologies, Grand Island, NY, USA) lysis buffer.
9
Isolation and Identification of Murine Hematopoietic Progenitors
10
Th1/Th2 Immune Response and N. brasiliensis Infection
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