Monocytes were isolated from umbilical cord blood by centrifugation using ficoll (TBD, Tianjin, China). Umbilical cord blood was retrieved from full-term healthy pregnancies during cesarean sections. Research approval had been previously retrieved from the ethics committee of Beijing Chao-Yang Hospital (2020-science-154), and informed consent was retrieved from all the patients involved. The clinical study was conducted following the provisions of the Declaration of Helsinki (as revised in 2013). Red blood cells were removed using red blood cells lysis buffer (Solarbio, Beijing, China). Monocytes were washed with PBS (Life Technologies) and separated using a magnetic activated cell sorting (MACS) column with anti-CD14-conjugated microbeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s protocol. CD14+ monocytes were cultured in Iscove’s modified Dulbecco’s medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% FBS (HyClone, Logan, UT, USA), 100 U/mL recombinant human granulocyte colony-stimulating factor (rhGM-CSF), and 50 U/mL rhIL-4. CD14− mononuclear cells were cultured in the same medium supplemented with rhIL-2 (20 ng/mL) to induce the immature DCs.
Anti cd14 conjugated microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
Anti-CD14-conjugated microbeads are a lab equipment product used for the isolation and enrichment of CD14-positive cells from various biological samples. The microbeads are coated with antibodies specific to the CD14 surface marker, which is expressed on monocytes and macrophages. This product can be used in cell separation and purification workflows.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by Thermo Fisher Scientific (4 mentions)
Interleukin 4 (il 4), by Miltenyi Biotec (2 mentions)
Gm csf, by Gentaur (2 mentions)
Hr granulocyte macrophage colony stimulating factor gmcsf, by R&D Systems (2 mentions)
Standard 5 μm pore filter, by Neuro Probe (2 mentions)
48 well boyden microchamber, by Neuro Probe (2 mentions)
Rosettesep human monocyte enrichment kits, by STEMCELL (2 mentions)
Cd4 cd127low t cell enrichment kits, by STEMCELL (2 mentions)
Facs aria iitm, by BD (2 mentions)
Ficoll paque plus, by Cytiva (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
Red blood cells lysis buffer, by Solarbio (1 mentions)
Iscove s modified dulbecco s medium, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharide, by InvivoGen (1 mentions)
Gibco s serum free aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Transforming growth factor (tgf), by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
21 protocols using anti cd14 conjugated microbeads
1
Isolation and Culture of Monocytes and DCs
2
Generation of M1, M2, and TAM-like Macrophages
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Heparinized leukocyte-enriched buffy coats were obtained from healthy blood donors, and peripheral blood mononuclear cells (PBMCs) were separated from buffy coats by Ficoll-Paque Plus (Biosciences) gradient centrifugation. Monocytes were purified from PBMCs by positive selection using immunomagnetic cell separation and anti-CD14-conjugated microbeads (Miltenyi Biotec), according to the manufacturer’s instructions. After separation on a VarioMACS magnet, 96–99% of the cells were shown to be CD14+ monocytes.
Isolated monocytes were cultured for 5 days in 6-well tissue culture plates at a density of 2.0 × 106 cells/ml in Gibco’s serum-free AIM-V medium (Thermo Fischer Scientific) supplemented with 50 ng/ml M-CSF (PeproTech). In order to acquire the M1 and M2 types, cells were stimulated on the fifth day of differentiation for 24 h with lipopolysaccharide (50 ng/ml ultrapure LPS, InvivoGen), IFNγ (20 ng/ml, PeproTech) to M1 and IL-4 (20 ng/ml, PeproTech), IL-10 (20 ng/ml, PeproTech) and TGFß (20 ng/ml, PeproTech) to M2 phenotype. For the differentiation of TAM-like cells, isolated monocytes were cultured for 5 days in 6-well tissue culture plates at a density of 2.0 × 106 cells/ml in Thp-1 supernatant supplemented with IL-4 (20 ng/ml), IL-10 (20 ng/ml) and TGFß (20 ng/ml). On the fifth day, TAM-like cells were treated again with Thp-1 supernatant for 24 h.
Isolated monocytes were cultured for 5 days in 6-well tissue culture plates at a density of 2.0 × 106 cells/ml in Gibco’s serum-free AIM-V medium (Thermo Fischer Scientific) supplemented with 50 ng/ml M-CSF (PeproTech). In order to acquire the M1 and M2 types, cells were stimulated on the fifth day of differentiation for 24 h with lipopolysaccharide (50 ng/ml ultrapure LPS, InvivoGen), IFNγ (20 ng/ml, PeproTech) to M1 and IL-4 (20 ng/ml, PeproTech), IL-10 (20 ng/ml, PeproTech) and TGFß (20 ng/ml, PeproTech) to M2 phenotype. For the differentiation of TAM-like cells, isolated monocytes were cultured for 5 days in 6-well tissue culture plates at a density of 2.0 × 106 cells/ml in Thp-1 supernatant supplemented with IL-4 (20 ng/ml), IL-10 (20 ng/ml) and TGFß (20 ng/ml). On the fifth day, TAM-like cells were treated again with Thp-1 supernatant for 24 h.
3
Isolation and Differentiation of Dendritic Cells
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Human peripheral blood mononuclear cells from healthy donors were isolated by Lympholyte cell separation media (Cedarlane, CL5020). Monocytes were isolated by immunomagnetic cell separation using anti-CD14-conjugated microbeads (Miltenyi Biotec, 1300-50-201). To induce the differentiation of immature DCs (iDCs), monocytes were cultured for 6 days with recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF)(50 ng/mL) (Milteny Biotec, 130-093-865) and interleukin-4 (IL-4) (20 ng/mL) (Miltenyi Biotec, 130-095-373).
4
Monocyte-Derived Dendritic Cell Generation
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Human PBMCs from healthy donors were isolated by Lymphoprep gradient centrifugation (Nycomed). Monocytes were isolated by immunomagnetic cell separation using anti-CD14-conjugated microbeads (Miltenyi Biotec, 1300-50-201). To induce the differentiation of iDCs, monocytes were cultured for 6 days with human recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) (50 ng/mL) (Milteny Biotec, 130-093-865) and interleukin-4 (IL-4) (20 ng/mL) (Miltenyi Biotec, 130-095-373), as previously described [58 (link)].
5
Monocyte Isolation and Differentiation
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Peripheral blood mononuclear cells were obtained from blood samples using Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden) gradient centrifugation. To purify the monocytes from the PMBCs, positive magnetic cell separation was performed using anti-CD14-conjugated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The isolated monocytes were plated at 1 × 106 cells/mL in RPMI-1640 medium (Merck KGaA, Darmstadt, Germany) supplemented with 10% FCS, 1% Penicillin-Streptomycin, and 1 g/L L-glutamine (all from Gibco, Thermo Fisher Scientific, Waltham, MA, USA). For the induction of differentiation, 100 ng/mL IL-4 (PeproTech EC, London, UK) and 80 ng/mL GM-CSF (Gentaur Molecular Products, Brussels, Belgium) were added to the cells.
6
Isolation and Differentiation of Human PBMCs
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Peripheral blood mononuclear cells (PBMCs) were isolated by buffy coats through gradient centrifugation using Ficoll-Paque (GE Healthcare Italia, cat #45-001-750), according to the manufacturer’s recommendations. CD14+-cells were isolated using anti-CD14 conjugated microbeads (Miltenyi Biotec, cat #130-050-201) [26 (link)]. Monocyte-derived dendritic cells (DCs) were obtained by stimulating CD14+ cells with recombinant IL-4 (50 ng/mL) and recombinant GM-CSF (100 ng/mL) [27 (link)].
CD4+ T-cells were isolated from a non-adherent fraction of PBMCs by using a CD4+ T-cells separation kit (Miltenyi, cat #130-096-533) according to the manufacturer’s recommendations. The purity of the populations was checked using cytofluorimetric analysis with specific antibodies and was always >90%.
CD4+ T-cells were isolated from a non-adherent fraction of PBMCs by using a CD4+ T-cells separation kit (Miltenyi, cat #130-096-533) according to the manufacturer’s recommendations. The purity of the populations was checked using cytofluorimetric analysis with specific antibodies and was always >90%.
7
Monocyte-Derived Cell Activation by Bacterial Exposure
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Buffy coats from 6 healthy donors were supplied by Transfusional Center of Azienda Ospedaliera Careggi (Firenze, Italy). CD14+ cells were isolated using anti-CD14 conjugated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). To obtain MDC CD14+ cells were cultured in the presence of 20 ng/ml of human recombinant (hr) IL-4 and 50 ng/ml of hr Granulocyte Macrophage Colony Stimulating Factor GMCSF (R&D Systems, Minneapolis, MN, USA) for seven days at 37°C in a humidified chamber with 5% CO2. MDC were recovered, plated at 106 cells/ml and stimulated with bacterial cells. Monocytes and MDC were cultured with live bacterial cells: 1:1 for 4–7 hours or with UV-inactivated bacterial cells 1:10 for the indicated times.
8
Monocyte-Derived Dendritic Cell Generation
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The use of buffy coats from donated blood, not usable for therapeutic purposes, was approved by the Ethics Committee of the Azienda Ospedaliera Universitaria Careggi in agreement with the D.M. of Italian Ministry of Health (15A09709) G.U., n. 300 12.28. 2015). Buffy coats were collected at the Transfusional Center of the Azienda Ospedaliera Universitaria Careggi (Firenze, Italy) and peripheral blood mononuclear cells (PBMC) were isolated by gradient centrifugation using Ficoll-Paque (GE Healthcare Italia, Milan, Italy), according to the manufacturer’s recommendations.
CD14+ cells were isolated using anti-CD14 conjugated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). To obtain monocyte-derived dendritic cells (MDC), CD14+ cells were cultured in the presence of 20 ng/ml of human recombinant (hr) IL-4 and 50 ng/ml of hr Granulocyte Macrophage Colony Stimulating Factor (GMCSF) (R&D Systems, Minneapolis, MN, USA) for seven days at 37°C in a humidified chamber with 5% CO2 [29 (link)]. MDC were recovered, plated at 106 cells/ml and stimulated with bacterial cells.
CD14+ cells were isolated using anti-CD14 conjugated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). To obtain monocyte-derived dendritic cells (MDC), CD14+ cells were cultured in the presence of 20 ng/ml of human recombinant (hr) IL-4 and 50 ng/ml of hr Granulocyte Macrophage Colony Stimulating Factor (GMCSF) (R&D Systems, Minneapolis, MN, USA) for seven days at 37°C in a humidified chamber with 5% CO2 [29 (link)]. MDC were recovered, plated at 106 cells/ml and stimulated with bacterial cells.
9
Monocyte Migration Assay using 9-GN
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Blood monocytes were purified from six donors by positive selection over a MACS column using anti-CD14-conjugated microbeads (Miltenyi Biotec) as previously described [60 (link)].
Monocyte migration (5 × 104 cells/well in 50 μL of RPMI 1640 medium with 0.1% FCS) was performed in a 48-well Boyden microchamber (Neuroprobe) through a standard 5-μm pore filter (Neuroprobe), at 37 °C for 1 h. Migration was evaluated in response to synthetic9-GN (1 to 100 nM in RPMI 1640 medium containing 0.1% FCS), CCL2 used as positive control (200 ng/mL, 23.1 nM, R&D Systems), and medium alone used as negative control. The filter was stained using Diff–Quik reagent. Migrated cells were counted on the lower side of the filter in three randomly selected high power fields (magnification × 100). Each assay was performed in triplicate. Results are expressed as the difference between mean numbers of cells per high power fields minus the negative control (medium alone).
Monocyte migration (5 × 104 cells/well in 50 μL of RPMI 1640 medium with 0.1% FCS) was performed in a 48-well Boyden microchamber (Neuroprobe) through a standard 5-μm pore filter (Neuroprobe), at 37 °C for 1 h. Migration was evaluated in response to synthetic
10
Monocyte-Derived Macrophage Differentiation
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Peripheral blood was collected from healthy volunteers from the Regional Blood Center of the Hungarian National Blood Transfusion Service (Debrecen, Hungary) with the approval of the Regional Institutional Research Ethics Committee of the University of Debrecen.
Human monocytes were separated as it was published previously (40 (link)). After density gradient centrifugation with Ficoll Plaque Plus (Amersham Biosciences; GE17-1440-02) the peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats. Positive selection of human monocytes from PBMCs was carried out using anti-CD14-conjugated MicroBeads (Miltenyi Biotec; 130-050-201) in accordance with the manufacturer’s protocol.
Isolated monocytes were suspended in RPMI 1640 (Sigma; D5671) media supplemented with 10% FBS and 5% penicillin/streptomycin/amphotericin b. In a 6-well cell culture plate 2x106 cells/ml were plated and placed in a humidified incubator at 37°C atmosphere containing 5% CO2 for 16 h before treatment. Adherent, monocyte-derived differentiating macrophages were treated with 20 ng/ml IL-4 (Peprotech; 200–04) under normoxic and hypoxic conditions with for the indicated period.
Human monocytes were separated as it was published previously (40 (link)). After density gradient centrifugation with Ficoll Plaque Plus (Amersham Biosciences; GE17-1440-02) the peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats. Positive selection of human monocytes from PBMCs was carried out using anti-CD14-conjugated MicroBeads (Miltenyi Biotec; 130-050-201) in accordance with the manufacturer’s protocol.
Isolated monocytes were suspended in RPMI 1640 (Sigma; D5671) media supplemented with 10% FBS and 5% penicillin/streptomycin/amphotericin b. In a 6-well cell culture plate 2x106 cells/ml were plated and placed in a humidified incubator at 37°C atmosphere containing 5% CO2 for 16 h before treatment. Adherent, monocyte-derived differentiating macrophages were treated with 20 ng/ml IL-4 (Peprotech; 200–04) under normoxic and hypoxic conditions with for the indicated period.
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