Anti cd14 magnetic microbeads

Manufactured by Miltenyi Biotec

Sourced in Germany

Anti-CD14 magnetic microbeads are a laboratory product designed for the enrichment or depletion of CD14-positive cells from various sample types. The microbeads are coated with antibodies specific to the CD14 cell surface marker, allowing for the magnetic separation of CD14-expressing cells from a heterogeneous cell population.

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Lab products found in correlation

Human il 4, by Immunotools (4 mentions) Human granulocyte macrophage colony stimulating factor, by Immunotools (3 mentions) Gm csf, by Miltenyi Biotec (2 mentions) Interleukin 4 (il 4), by Miltenyi Biotec (2 mentions) Defined hyclone fbs, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Immunotools (2 mentions) Ls separation column, by Miltenyi Biotec (1 mentions) Ficoll, by Merck Group (1 mentions) Human ab serum, by Merck Group (1 mentions) Il 1β, by CellGenix (1 mentions) Interleukin 6 (il 6), by CellGenix (1 mentions) Tnf α, by CellGenix (1 mentions) X vivo 15, by Lonza (1 mentions) Easysep human myeloid dc enrichment kit, by STEMCELL (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Merck Group (1 mentions) Penicillin, by Merck Group (1 mentions) Streptomycin, by Merck Group (1 mentions) L glutamine, by Merck Group (1 mentions) Lymphoprep, by Axis-Shield (1 mentions)

Close spelling variants of this product

CD14 MicroBeads (570 citations) Magnetic microbeads (147 citations) Anti-CD14 microbeads (121 citations) CD14 magnetic microbeads (47 citations) Anti-CD14-conjugated magnetic microbeads (22 citations) Anti-CD14 antibody-conjugated magnetic microbeads (5 citations) Anti-CD14 monoclonal antibodies conjugated to magnetic microbeads (3 citations) Anti-CD14-coated magnetic microbeads (3 citations) Anti-CD14 monoclonal antibody-coated magnetic microbeads (3 citations) Anti-CD14 mAb-conjugated magnetic microbeads (2 citations)

16 protocols using anti cd14 magnetic microbeads

Monocyte-derived dendritic cell protocol

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CD14+ cells within PBMCs were isolated using anti-CD14 magnetic microbeads and LS column separation (Miltenyi Biotech) per the manufacturer’s instructions. These cells were then cultured in complete RPMI media (10% FBS and 2 mM L-Glutamax) for 7 days in the presence of GM-CSF (20 ng/mL) and IL-6 (20 ng/mL). GM-CSF was added on days 1, 3, and 5, whereas IL-6 was added only on day 533 34 (link) (online supplemental figure 1). The cells were then harvested on day 7, and flow cytometry was performed to assess the phenotype (CD33⁺CD11b⁺HLA-DR^lowCD14⁺CD15^-).

Nalawade S.A., Shafer P., Bajgain P., McKenna M.K., Ali A., Kelly L., Joubert J., Gottschalk S., Watanabe N., Leen A., Parihar R., Vera Valdes J.F, & Hoyos V. (2021). Selectively targeting myeloid-derived suppressor cells through TRAIL receptor 2 to enhance the efficacy of CAR T cell therapy for treatment of breast cancer. Journal for Immunotherapy of Cancer, 9(11), e003237.

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Isolation and Culture of PBMCs and moDCs

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The samples from the controls were processed following the current procedures immediately after their reception. These were kindly provided by BBSSPA, unit HH.UU, Virgen Macarena Virgen del Rocío. PBMCs and moDCs were obtained from peripheral blood (40 mL) from the healthy subjects included in the study. PBMCs were isolated by centrifugation on a gradient using Ficoll (Sigma, Madrid, Spain) with standardized protocols. Monocytes were purified from the PBMCs by a positive selection with anti-CD14 magnetic microbeads following the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). These had a purity greater than 95%, as assessed by flow cytometry CD14⁻ cell fractions were frozen for further use. Monocytes (CD14⁺ cells) were cultured in a complete medium as previously described by us for the generation of monocyte-derived DCs [19 (link)].

Fernandez-Prades L., Brasal-Prieto M., Alba G., Martin V., Montserrat-de la Paz S., Cejudo-Guillen M., Santa-Maria C., Dakhaoui H., Granados B., Sobrino F., Palomares F, & Lopez-Enriquez S. (2023). Sulforaphane Reduces the Chronic Inflammatory Immune Response of Human Dendritic Cells. Nutrients, 15(15), 3405.

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Dendritic Cell Differentiation from Monocytes

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Mature dendritic cells (mDC) were derived, as reported previously [34] (link), from peripheral blood samples. Buffy coats from healthy donors were obtained from Banc de Sang I Teixits upon written informed consent. In brief, peripheral blood mononuclear cells (PBMCs) were allowed to adhere to a plastic surface for 2 h at 37°C. Unbound PBMCs were washed away, and the remaining adherent monocytes were cultured for 5 days in a 37°C, 95% humidity, 5% CO₂ incubator in the presence of IL-4 (300 U/ml) and GM-CSF (450 U/ml) (both from Miltenyi Biotec, Madrid, Spain) in X-VIVO 15 (BioWhittaker, Lonza Belgium) medium supplemented with 2% AB human serum (Sigma-Aldrich, Spain). After 5 days, DCs were matured for 48 hours using a cocktail of IL-1β, IL-6 (both at 1000 IU/ml), TNF-α (500 IU/ml) (all 3 from CellGenix, Freiburg, Germany) and Prostaglandin E2 (PGE2, 10 µg/ml; Dinoprostona, Pfizer). mDCs were harvested and brought to the proper concentration for subsequent experiments.
Monocytes in solution were positively selected from PBMCs using anti-CD14 magnetic microbeads (Miltenyi Biotec, Madrid, Spain).

Borgman K.J., van Zanten T.S., Manzo C., Cabezón R., Cambi A., Benítez-Ribas D, & Garcia-Parajo M.F. (2014). Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells. PLoS ONE, 9(6), e99589.

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Monocyte-derived Dendritic Cell Culture

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All DC cultures were carried out at 37°C, 5% CO₂, in RPMI-1640 (Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco BRL), 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (all Sigma-Aldrich). The use of Leukocyte Reduction System (LRS) cones from platelet donations from healthy volunteers was approved by the Newcastle and North Tyneside Research Ethics Committee 2. Peripheral blood mononuclear cells (PBMC) were isolated from LRS cones by density gradient centrifugation on Lymphoprep (Axis-Shield Diagnostics). To generate immature monocyte-derived DC (moDC) as previously described (23 (link), 24 ), CD14⁺ monocytes were isolated from PBMC by positive magnetic selection using anti-CD14 magnetic microbeads (Miltenyi Biotec) and cultured at 0.5 × 10⁶ cells/ml in a 24-well plate in the presence of IL-4 and GM-CSF (50 ng/ml each, Immunotools, Friesoythe, Germany) for 6 days. Medium supplemented with cytokines was refreshed at day 3. CD1c⁺ DC were separated from PBMC using an immunomagnetic negative selection kit (EasySep human myeloid DC enrichment kit; StemCell) and were cultured in a 96-well plate at 6.5 × 10⁴ cells/200 μl.

Maney N.J., Reynolds G., Krippner-Heidenreich A, & Hilkens C.M. (2014). Dendritic cell maturation and survival are differentially regulated by TNF receptors 1 and 2. Journal of immunology (Baltimore, Md. : 1950), 193(10), 4914-4923.

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Monocyte Isolation and LPS Stimulation

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CD14+ monocytes from NOMID patients or healthy donors were separated from PBMCs using anti-CD14 magnetic microbeads (Miltenyi Biotec, Auburn, CA), and an AutoMACS sorter (Miltenyi Biotec). Sorted monocytes were then stained with Qdot 655 anti-CD14, (clone (TüK4) (Invitrogen) and cultured in Lab-Tek chambered coverglass (Thermo Scientific, Waltham, MA) in complete medium. Cells were stimulated with 50 ng/mL LPS (Sigma, St. Louis, MS) and observed with a Zeiss LSM 510 Meta microscope (Carl Zeiss Microscopy, LLC Thornwood, NY). Pictures were merged using ImageJ software (National Institutes of Health, Bethesda, MD).

Edwan J.H., Goldbach-Mansky R, & Colbert R.A. (2015). Identification of IL-1β-producing monocytes that are susceptible to pyronecrotic cell death in patients with NOMID. Arthritis & rheumatology (Hoboken, N.J.), 67(12), 3286-3297.

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Differentiation of THP1 and CD14+ Monocytes

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THP1 cells, a human acute monocytic cell line that was purchased from the Human Science Foundation (Tokyo, Japan), were cultured in RPMI-1640 supplemented with 10% HI-FBS (Thermo Fisher Scientific, Waltham, MA, USA). Differentiation was achieved by re-suspending the cells at 8 × 10⁵ cells/ml in Dulbecco’s modified Eagle’s medium (Wako) supplemented with 10% heat-inactivated fetal bovine serum and penicillin-streptomycin with the addition of 100 nM phorbol 12-myristate 13-acetate (Sigma) for 48 hours. Non-adherent cells were removed by aspiration of the supernatant followed by replacement with fresh medium.
The human blood PBMC was obtained by using Optiprep (Axis-Shield, Dundee, Scotland) in accordance with the manufacturer’s instructions. CD14⁺ monocytes were then isolated from PBMC by the AutoMacs separation system using anti-CD14 magnetic microbeads (Miltenyi Biotec, Germany). The CD14⁺ human monocytes were differentiated to macrophages by commercially available macrophage differentiation kit (R&D systems, Minnesota).

Inoue M., Niki M., Ozeki Y., Nagi S., Chadeka E.A., Yamaguchi T., Osada-Oka M., Ono K., Oda T., Mwende F., Kaneko Y., Matsumoto M., Kaneko S., Ichinose Y., Njenga S.M., Hamano S, & Matsumoto S. (2018). High-density lipoprotein suppresses tumor necrosis factor alpha production by mycobacteria-infected human macrophages. Scientific Reports, 8, 6736.

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Isolation of Healthy Control PBMCs

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Blood from healthy controls (HC) was obtained following institutional ethical approval. Peripheral blood mononuclear cells (PBMCs) from heparinized blood were isolated by density centrifugation using Ficoll-Paque Plus (GE Healthcare). Fresh monocytes were isolated using anti-CD14 magnetic microbeads (Miltenyi Biotec) based on positive separation on auto-MACS assisted cell sorting (Miltenyi Biotec) according to the manufacturer's protocol.

Carvalheiro T., Garcia S., Pascoal Ramos M.I., Giovannone B., Radstake T.R., Marut W, & Meyaard L. (2020). Leukocyte Associated Immunoglobulin Like Receptor 1 Regulation and Function on Monocytes and Dendritic Cells During Inflammation. Frontiers in Immunology, 11, 1793.

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Isolation and Culture of Human Immune Cells

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Human PBMCs were separated using Ficoll-Paque (GE Healthcare, Uppsala, Sweden) density gradient centrifugation from whole blood obtained from Kraft Family Blood Donor Center, Boston, MA according to an Institutional Review Board approved protocol. CD4+ T cells were separated from the CD14-negative fraction of PBMCs using CD14 and CD4 magnetic microbeads (MACS Miltenyi Biotec, Auburn, CA). T cells were cultured in RPMI 1640 media (Cellgro, Manassas, VA) containing 10% Human Serum (AB) (GemCell, West Sacramento, CA), 100 U/ml penicillin and streptomycin sulfate 100 μg/ml (H10 medium) supplemented with 5 ng/ml rhIL-15 (R&D Systems, Minneapolis, MN) to maintain cell viability without cell activation. Human Monocyte derived Dendritic Cells (MDDCs) were prepared from CD14-positive monocytes selected from peripheral blood mononuclear cells using anti-CD14 magnetic microbeads (MACS Miltenyi Biotec) and cultured for 6 days with 100 ng/ml interleukin-4 and 50 ng/ml granulocyte-macrophage colony-stimulating factor (R & D Systems).

Sharei A., Trifonova R., Jhunjhunwala S., Hartoularos G.C., Eyerman A.T., Lytton-Jean A., Angin M., Sharma S., Poceviciute R., Mao S., Heimann M., Liu S., Talkar T., Khan O.F., Addo M., von Andrian U.H., Anderson D.G., Langer R., Lieberman J, & Jensen K.F. (2015). Ex Vivo Cytosolic Delivery of Functional Macromolecules to Immune Cells. PLoS ONE, 10(4), e0118803.

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Human Monocyte-Derived Dendritic Cell Generation

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Human monocyte-derived immature dendritic cells were generated from peripheral blood mononuclear cells (PBMCs) obtained from buffy coat preparations (National Blood Centre, St. James’s Hospital, Dublin) by density gradient centrifugation (Lymphoprep) as described^{43 (link),48 (link)}. Briefly, monocytes were isolated by positive selection using anti-CD14 magnetic microbeads, as described by the manufacturer (Miltenyi Biotec) and seeded at a density of 1 × 10⁶ cells/mL in 6-well plates in 3 mL of Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco) containing 10% defined HyClone FBS (Thermo Scientific), 1% penicillin/streptomycin (Lonza, Basel, Switzerland), 1% fungizone (Sigma-Aldrich), human granulocyte macrophage colony-stimulating factor (GM-CSF, 50 ng/mL; Immunotools, Germany), and human IL-4 (70 ng/mL; Immunotools) in a humidified atmosphere with 5% CO₂ at 37 °C. Cells were fed at day 3 by replacing half the medium with fresh cytokine-supplemented RPMI. At day 6, cells exhibited a CD11c⁺ immature dendritic cell phenotype.

Buckley A.M., Dunne M.R., Morrissey M.E., Kennedy S.A., Nolan A., Davern M., Foley E.K., Clarke N., Lysaght J., Ravi N., O’Toole D., MacCarthy F., Reynolds J.V., Kennedy B.N, & O’Sullivan J. (2020). Real-time metabolic profiling of oesophageal tumours reveals an altered metabolic phenotype to different oxygen tensions and to treatment with Pyrazinib. Scientific Reports, 10, 12105.

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Generation of Immature Dendritic Cells

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Human monocyte-derived immature DCs were generated from peripheral blood mononuclear cells (PBMCs) obtained from buffy coat preparations (National Blood Centre, St. James’s Hospital, Dublin) by density gradient centrifugation (Lymphoprep) as we previously described (Supplementary Fig. 1 A) [18 (link), 43 (link)]. Briefly, monocytes were isolated by positive selection using anti-CD14 magnetic microbeads as described by the manufacturer (Miltenyi Biotec) and seeded at a density of 1 × 10⁶ cells/mL in 6-well plates in 3 mL of RPMI-1640 medium containing 10% defined HyClone FBS (Thermo Scientific), 1% penicillin/streptomycin, 1% fungizone, human granulocyte macrophage colony-stimulating factor (50 ng/mL; Immunotools), and human IL-4 (70 ng/mL; Immunotools) in a humidified atmosphere with 5% CO₂ at 37 °C. Cells were fed at day 3 by replacing half the medium made up with fresh cytokines as above. At day 6, CD11c⁺ cells exhibited an immature DC phenotype capable of upregulating cell surface markers following LPS activation.

Morrissey M.E., Byrne R., Nulty C., McCabe N.H., Lynam-Lennon N., Butler C.T., Kennedy S., O’Toole D., Larkin J., McCormick P., Mehigan B., Cathcart M.C., Lysaght J., Reynolds J.V., Ryan E.J., Dunne M.R, & O’Sullivan J. (2020). The tumour microenvironment of the upper and lower gastrointestinal tract differentially influences dendritic cell maturation. BMC Cancer, 20, 566.

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