After mice were sacrificed, small intestines were immediately placed in cold PBS. Fat and Peyer’s Patches were removed and the intestines were rinsed with cold PBS. After longitudinally opening of the intestines, they were cut into small pieces and put into 20 mL HBSS (Sigma, St. Louis, MI, USA) supplemented with 5% FSC (Pan Biotech, Aidenbach, Germany), 10 mM HEPES (Sigma, St. Louis, MI, USA), 5 mM EDTA and 1 mM Dithiothreitol (Sigma, St. Louis, MI, USA). The pieces were incubated at 37 °C for 20 min, vortexed for 15 s, and transferred onto a 100 µm filter. The flowthrough contained the IELs. After repeating this step once, gut pieces were incubated in HBSS supplemented with 10 mM HEPES for 20 min, vortexed, and also put on a 100 µm filter. The flowthrough was spun down and transferred to a density gradient with 70% and 40% Percoll (GE Healthcare, Chicago, IL, USA) to separate the IEL-fraction. Afterward, lymphocytes were enriched by magnetical depletion with anti-EpCAM-, anti CD11c- and anti CD11b beads (all Miltenyi Biotech, Bergisch Gladbach, Germany). Purity was checked by flow cytometry and was routinely at a ratio of 20% CD4+ and 80% CD8+.
Anti cd11b beads
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD11b beads are a type of magnetic separation beads from Miltenyi Biotec. They are used for the isolation and enrichment of CD11b-positive cells from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Anti ly6g microbead kit, by Miltenyi Biotec (1 mentions)
Macs bead, by Miltenyi Biotec (1 mentions)
Magnetic ms column, by Miltenyi Biotec (1 mentions)
Dead cell removal kit, by Miltenyi Biotec (1 mentions)
5 protocols using anti cd11b beads
1
Isolation of Intestinal Intraepithelial Lymphocytes
2
Isolation of CD11b+ Lamina Propria Cells
3
Purification of Neutrophils and Inflammatory Monocytes
4
Adoptive Transfer of Myeloid Cells for EAE
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To test the therapeutic role of CNS-infiltrating myeloid cells, healthy B6 mice or mice showing first symptoms of EAE received intrathecal MIS416 (100 μg). CNS tissues were isolated from donor mice 1 day post injection and prepared for cell sorting to obtain monocytic (CD45hiCD11bhiGR-1low/−F4/80+) and granulocytic (CD45hiCD11bhiGR-1hiF4/80−) cell populations. To test the therapeutic role of peripherally induced myeloid cells, spleens were isolated 1 day post injection from animals which received MIS416 intravenously (iv), and mechanically dissociated. Red cell lysis buffer 0.83% NH4CL (Merck, Germany) was used to remove erythrocytes. A pre-sort using anti-CD11b beads (Miltenyi Biotech) was performed to enrich the percentage of myeloid fraction. The positive fraction was then stained with fluorochrome-conjugated antibodies to identify neutrophils (CD11bhiLy6G+Ly6CdimCD11c−F4/80−) and monocytes (CD11bhiLy6G−Ly6Cdim/hiCD11c−F4/80low). Selected myeloid cells were then sorted and injected intrathecally to recipient mice showing first symptoms of EAE. Mice received 1,5–2,0 × 105 cells/mouse. Mice treated with sorted cells were observed over the following 4 days to assess disease progress.
5
Isolation of CD11b+ Tumor Infiltrating Cells
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CD11b+ cells were isolated from tumors by using MACS beads (Miltenyi, Bergisch-Gladbach, Germany), as previously reported.12 (link) Briefly, tumors were incubated with collagenase 4 and DNase I for 20 min at 37°C, followed by mechanical disruption. After single-cell suspensions were obtained, dead cells were depleted by using a dead cell removal kit from Miltenyi according to the manufacturer's protocol. Thereafter, aliquots of 107 cells were incubated for 15 min with 10 μL of anti-CD11b beads (clone M1/70.15.11.5) in 100 μL MACS buffer (PBS, 0.5 mmol EDTA, 0.5% BSA) at 4°C, washed two times with MACS buffer and subjected to two consecutive rounds of separation via magnetic MS columns (Miltenyi). This procedure yielded predominantly CD11b+ cells with purity greater than 80% as assessed by FACS analysis.
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