Splenocytes from non-tumor bearing and 4T1-bearing mice (tumor volume >1000 mm3) were depleted of CD11b+ cells using anti-CD11b+ magnetic beads (<5% residual CD11b+Gr-1+; Miltenyi Biotec). CD11b+-depleted splenic cell populations were then labeled either with CellTrace CFSE or CellTrace Violet (ThermoFisher), respectively and co-mixed at a 1:1 ratio. For in vitro studies, the co-mixture was cultured at a concentration of 106 cells ml−1 in complete media supplemented with 1 mM sodium pyruvate, MEM non-essential amino acids, and 25 mM HEPES. For in vivo recovery studies, 2–3 × 107 cells of the co-mixture were adoptively transferred via the tail vein into non-tumor bearing recipients. L-selectin expression on CD4+ and CD8+ T cells was determined by flow cytometric analysis prior to transfer (input) or four days after culture or adoptive transfer into mice.
Anti cd11b magnetic beads
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD11b magnetic beads are a laboratory product designed for cell separation and purification. These beads are coated with antibodies that specifically bind to the CD11b antigen expressed on the surface of certain cell types. The magnetic properties of the beads allow for the efficient isolation and enrichment of CD11b-positive cells from complex samples, facilitating further analysis or downstream applications.
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Lab products found in correlation
Neural tissue dissociation kit, by Miltenyi Biotec (6 mentions)
Ms column, by Miltenyi Biotec (4 mentions)
Percoll plus, by GE Healthcare (3 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Celltrace cfse, by Thermo Fisher Scientific (1 mentions)
Magnetic separation column, by Miltenyi Biotec (1 mentions)
Penicillin streptomycin, by Harvard Bioscience (1 mentions)
Tumor necrosis factor (tnf), by Miltenyi Biotec (1 mentions)
Liberase dl, by Roche (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Neural tissue dissociation kit p, by Miltenyi Biotec (1 mentions)
Celltracker orange, by Thermo Fisher Scientific (1 mentions)
Calcein, by Thermo Fisher Scientific (1 mentions)
14 protocols using anti cd11b magnetic beads
1
Adoptive Transfer of Depleted Splenocytes
2
Acute Microglia Isolation from Mouse Brain
3
Rapid Microglia Isolation via MACS
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Microglia isolation using Miltenyi CD11b‐conjugated magnetic microbeads was described previously (Chen et al., 2018 ). Briefly, brains were quickly dissociated enzymatically with a Neural Tissue Dissociation Kit (Miltenyi Biotec). Microglia were subsequently purified by magnetic‐activated cell sorting (MACS) using anti‐CD11b magnetic beads (Miltenyi Biotec). The whole process took about 60–90 min and isolated cells were immediately used for downstream applications without culturing or exposure to serum.
4
In Vivo Prenylation of Rab Proteins
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Studies with mice were approved by the Garvan Institute of Medical Research/St Vincent's Hospital Animal Ethics Committee. Adult Balb/c mice were untreated or injected with a single dose of 100 μg/kg ZOL either s.c. in the scruff of the neck, i.p. or i.v. via the tail vein (3 mice per group). In a separate study, 2 mice were injected s.c. once per week for 8 weeks with ZOL or phosphate-buffered saline (2 mice per group). Mice were euthanased 24 h after the final injection, then peritoneal cells (already approximately 40% F4/80+ macrophages) were harvested by lavage. CD11b+ macrophages were further purified using Miltenyi anti-CD11b magnetic beads according to the manufacturer's protocol. The purified macrophages from each mouse were lysed in IVP buffer, then 50 μg of lysate were used for IVP reactions with Rab GGTase as described above and analyzed for in vitro-prenylated (biotinylated) Rab proteins and for unprenylated Rap1A by hybridizing blots with streptavidin-680 RD, followed by anti-unprenylated Rap1A and anti-goat-800 CW antibodies. For label-free MS analysis, 70 μg of lysate were made up to a total of 80 µl with IVP buffer containing 2 µM RabGGTase, 2 µM REP1, 1 µM B-GPP, then in vitro-prenylated proteins were enriched with magnetic streptavidin beads and analyzed by nano-LC-MS as described below.
5
Isolation and Characterization of Murine Splenocytes
6
Isolation of Microglia and Macrophages
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For Western blot analysis, microglia and macrophages were separated from a heterogeneous mixture of cells (i.e. inflammatory cells, glia, and neurons) using MACS. The single-cell suspension obtained from spinal tissue was incubated for 15 min with anti-CD11b magnetic beads (Miltenyi Biotec, Germany). After incubation, the suspension was rinsed with 10% BSA to remove unattached CD11b beads. The cells were resuspended in 3 mL of 10% BSA and passed through magnetic separation columns (Miltenyi Biotec, Germany) where CD11b+ cells were trapped while all other cells flowed freely out of the column. Unlabeled cells were collected as they exited the column as the “negative fraction”. The “positive fraction” or CD11b+ cells were extracted from the column by removing the magnetic separation column from the magnetic stand and flushing the cells out using the supplied plunger and 10% BSA.
7
Isolation and TNF-induced Stimulation of Murine MSCs
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Mesenchymal stem cells were isolated from femora and tibiae of Thy-1-deficient and WT mice by enzymatic digestion using 26 U/ml of Liberase DL (Roche) for 2 h at 37°C and 5% CO2. Cell suspension was filtered through a cell strainer (70 μm) and cultured in a-MEM medium (Lonza) supplemented with 10% fetal calf serum (FCS) (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Biochrom AG) at 37°C, 5% CO2. CD11b-positive cells were removed by magnetic cell separation using anti-CD11b magnetic beads according to the manufacturer’s protocol (Miltenyi). Purity was checked as described previously (Picke et al., 2018a (link)). MSCs were stimulated with 10 ng/ml TNF (Miltenyi) for 24 h.
8
Isolating Microglia from Mouse Brain
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Animals were euthanized via CO2 asphyxiation and transcardial perfusion with sterile ice-cold saline. Brain tissue (all but the hippocampus which was frozen in dry ice) was collected and used immediately for microglia isolation using a procedure adapted from Nikademova and Watters (27 (link)). Brains were enzymatically digested using the Neural Tissue Dissociation Kit (Miltenyi Biotec, Germany) for 35 min at 37°C. Further processing was performed at 4°C. Tissue debris was removed by passing the cell suspension through a 40-µm cell strainer. After myelin removal using 30% Percoll Plus (GE Healthcare, Princeton, NJ, USA), cells in PBS supplemented with 0.5% BSA and 2 mM EDTA were incubated for 15 min with anti-Cd11b magnetic beads (Miltenyi Biotec, Germany). CD11b+ cells were extensively washed and separated in a magnetic field using MS columns (Miltenyi Biotec, Germany) before being directly placed in Trizol reagent (Invitrogen).
9
Isolation of Murine Microglia from Brain Tissue
10
Rapid Isolation of Adult Microglia
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