E0771-GFP tumor cells were plated either alone or in direct contact with BAC1.2F5 macrophages at a 1:4 ratio (200,000 tumor cells and 800,000 macrophages) for 48 h in DMEM supplemented with 10% FBS. The following day, tumor cells and macrophages were collected and tumor cells were isolated from macrophages using MACS CD11b Microbeads (Miltenyi Biotec, cat #130-049-601), as previously described101 (link). Briefly, after counting, 1 × 107 cells were resuspended in 90 µL of buffer (PBS, 0.5% BSA, and 2 mM EDTA) and 10 µL of MACS CD11b Microbeads (Miltenyi Biotec). These microbeads are colloidal super-paramagnetic beads conjugated with monoclonal anti-mouse CD11b (Mac-1α) antibodies. The cells were placed at 4 °C for 15 min and then placed in a magnetic separator to separate macrophages from tumor cells. The purification of tumor cells from macrophages is in a range of 95%, as we previously described101 (link). Collected tumor cells were counted immediately and intravenously injected in mice bearing a lung window.
Macs cd11b microbead
Manufactured by Miltenyi Biotec
Sourced in Germany
MACS CD11b microbeads are magnetic beads conjugated with an antibody that specifically binds to the CD11b surface antigen. They are used for the isolation and enrichment of CD11b-positive cells from a heterogeneous cell population.
Automatically generated - may contain errors
Lab products found in correlation
Pen strep, by Thermo Fisher Scientific (4 mentions)
Anti cd11b microbead, by Miltenyi Biotec (2 mentions)
Macs myelin removal beads 2, by Miltenyi Biotec (2 mentions)
Cd11b microbeads, by Thermo Fisher Scientific (2 mentions)
Neural tissue dissociation kit p, by Miltenyi Biotec (2 mentions)
Dmem glutamax medium, by Thermo Fisher Scientific (2 mentions)
Ms column, by Miltenyi Biotec (2 mentions)
Fetal bovine serum (fbs), by Biosera (2 mentions)
Ficoll density gradient centrifugation, by Cytiva (1 mentions)
Iscript reverse transcription supermix, by Bio-Rad (1 mentions)
Taqman primer, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Adult brain dissociation kit, by Miltenyi Biotec (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
40 μm nylon mesh, by BD (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Raw264.7 cell line, by American Type Culture Collection (1 mentions)
Granulocyte macrophage colony stimulating factor gm csf, by Thermo Fisher Scientific (1 mentions)
7 protocols using macs cd11b microbead
1
Tumor Cell Isolation from Macrophages
2
Isolation of CD11b+ Myeloid Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from healthy anonymized blood donors’ buffy coat (Karolinska University Hospital) by Ficoll density gradient centrifugation (Cytiva). CD11b+ myeloid cells were isolated using MACS CD11b MicroBead (Miltenyi Biotec) according to manufacturer’s instruction.
3
Brain Tissue Extraction and RNA Isolation
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HsTX1[R14A] was purchased from Peptides International (Louisville, KY). RNeasy Lipid Tissue Mini Kit and RNeasy Micro Kit were purchased from Qiagen (Hilden, Germany). MACS® CD11b MicroBead and Adult Brain Dissociation Kit were purchased from MiltenyiBiotec (North Rhine-Westphalia, Germany). Phosphate-buffered saline (PBS) and Taqman™ primers were sourced from ThermoFisher Scientific (Waltham, MA). iScript™ Reverse Transcription Supermix was purchased from Bio-Rad (Hercules, CA).
4
Isolation and culture of primary murine microglia
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Microglia cells from either newborn (P0-1, male or female) or adult (P40-55, male) C57BL/6J, NKCC1fl/fl (WT), or NKCC1fl/fl ΔCx3CR1 (KO) mice were isolated by magnetic separation using anti-CD11b microbeads (Miltenyi Biotec, Germany), with slight modification of the protocol described by Otxoa-de-Amezaga [85 (link)]. After transcardial perfusion with ice-cold phosphate-buffered saline (PBS), the brain tissues (cortices and hippocampi) were enzymatically dissociated with Neural Tissue Dissociation Kit-P (#130-092-628; Miltenyi Biotec). Myelin was removed by MACS Myelin Removal Beads II (#130-096-733, Miltenyi Biotec), then cells in a single-cell suspension were magnetically labeled with MACS CD11b microbeads (#130-093-634, Miltenyi Biotec) and were separated using MS columns (#130-042-201, Miltenyi Biotec, Germany). Cells selected with CD11b microbeads were plated onto poly-L-lysine precoated 96-well or 386-well plates at 3 × 104 cell/cm2 density and were cultured at 37°C in a 95% air/5% CO2 incubator in DMEM/Glutamax medium (#31966–021, Gibco) supplemented with 10% fetal bovine serum (FBS, #FB-1090, Biosera), 1% Pen/Strep (10,000 U/ml; #15140–122, ThermoFisher Scientific), and 10 nM macrophage colony-stimulating factor (M-CSF; #PMC2044, ThermoFisher Scientific) for 10 days.
5
Isolation and Co-culture of Spleen-Derived Macrophages
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C57BL/6 mice splenocytes were harvested and washed in ice-cold Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco-BRL, Gaithersburg, MD, USA). The tissue was triturated using sterile syringes, and the resulting cell suspension was filtered via a 40-μm nylon mesh (BD Biosciences, North Ryde, Australia) and then incubated at 37°C for 30 minutes. The adherent cells were harvested and purified using MACS CD11b+ MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). These spleen-derived macrophages were rinsed three times with RPMI 1640 medium and cultured ex vivo with MSCs.
6
Isolation and Culture of Mouse Dendritic Cells
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To isolate DCs, monocytes were isolated from the bone marrow of C57BL/6 mice, which were then cultured in RPMI 1640 medium (Biowest, USA) supplemented with 10% fetal bovine serum, 50 U/mL penicillin/streptomycin, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 2 mmol/L nonessential amino acids, 10 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (Peprotech), and 5 ng/mL IL4 (Peprotech) at 37 °C and 5% CO2. The monocytes were incubated for 6 days before use in the experiment. The murine macrophage RAW 264.7 cell line was purchased from ATCC and cultured in DMEM supplemented with 10% fetal bovine serum and 50 U/mL penicillin/streptomycin. The cells were cultured at 37 °C in a humidified atmosphere cultivator with 5% CO2. To isolate peritoneal resident macrophages, peritoneal cells were collected by washing the peritoneal cavity with 10 mL of 1× phosphate-buffered saline (PBS) and then centrifuged. Pellets were resuspended with RBC lysis buffer and washed twice using 1× PBS. The acquired cells were sorted according to the MACS CD11b MicroBeads protocol (Miltenyi Biotec, Germany) and cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum and 50 U/mL penicillin/streptomycin.
7
Isolation and Culture of Mouse Microglia
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Microglia cells from either newborn (P0-1, male or female) or adult (P40-55, male) C57BL/6J, NKCC1 fl/fl (WT) or NKCC1 fl/fl Δ Cx3CR1 (KO) mice were isolated by magnetic separation using anti-CD11b microbeads (Miltenyi Biotec, Germany), with slight modification of the protocol described by Otxoa-de-Amezaga [81] (link). After transcardial perfusion with ice-cold PBS (phosphate buffered saline), the brain tissues (cortices and hippocampi) were enzymatically dissociated with Neural Tissue Dissociation Kit-P (#130-092-628; Miltenyi Biotec). Myelin was removed by MACS Myelin Removal Beads II (#130-096-733, Miltenyi Biotec), then cells in a single-cell suspension were magnetically labeled with MACS CD11b microbeads (#130-093-634, Miltenyi Biotec) and were separated using MS columns (#130-042-201, Miltenyi Biotec, Germany). Cells selected with CD11b microbeads were plated onto poly-L-lysine precoated 96-well or 386-well plates at 3x10 4 cell/cm 2 density and were cultured at 37 °C in a 95% air/5% CO2 incubator in DMEM/Glutamax medium (#31966-021, Gibco) supplemented with 10% FBS (fetal bovine serum, #FB-1090, Biosera), 1% Pen/Strep (10000 U/ml; #15140-122, ThermoFisher Scientific) and 10 nM Macrophage Colony-Stimulating Factor (M-CSF; #PMC2044, ThermoFisher Scientific) for 10 days.
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