Anti cd14 antibody

Manufactured by BD

Sourced in United States

The Anti-CD14 antibody is a laboratory reagent used in various immunological research applications. It binds specifically to the CD14 cell surface receptor, which is expressed on monocytes, macrophages, and other immune cells. The primary function of this antibody is to facilitate the detection and analysis of CD14-positive cells in samples.

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Lab products found in correlation

Ficoll paque plus, by GE Healthcare (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Phorbol myristate acetate (pma), by Merck Group (2 mentions) Recombinant human il 4, by R&D Systems (1 mentions) Rpmi medium, by Thermo Fisher Scientific (1 mentions) G csf, by BioLegend (1 mentions) Human m csf, by BioLegend (1 mentions) Heat inactivated human ab serum, by Thermo Fisher Scientific (1 mentions) Facscanto 2, by BD (1 mentions) Flow cytometry, by BD (1 mentions) Direct fluorescein isothiocyanate labeled anti cd11b antibody, by BD (1 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions) Facsaria 3 cell sorter, by BD (1 mentions) Ficoll paque plus, by Cytiva (1 mentions) Dznep, by Selleck Chemicals (1 mentions) Gsk126, by Selleck Chemicals (1 mentions) Lps escherichia coli 0111 b4, by Merck Group (1 mentions) Uplps, by InvivoGen (1 mentions) Gm csf, by R&D Systems (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

CD14 antibody (3 citations) PE-conjugated anti-CD14 antibody (3 citations) FITC-conjugated anti-human CD14 antibody (3 citations) Anti-CD14-FITC antibody (2 citations) PhycoErytherin (PE)-anti-human CD14 antibody (2 citations) Anti-human CD14 antibody (2 citations) Anti-CD14 monoclonal antibody (2 citations) Mouse anti-human CD14 antibody (2 citations) APC anti-human CD14 antibody (2 citations) Anti-human CD14-Percp-Cyc5.5 antibody (2 citations)

14 protocols using anti cd14 antibody

Monocyte Differentiation into Macrophages

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Peripheral blood mononuclear cells were isolated from buffy coats from healthy donors (Blutspende Zürich, Zurich, Switzerland) by density gradient centrifugation using Ficoll-paque PLUS (Cat: 17–1440–02; GE Healthcare). The monocyte population was magnetically enriched by negative selection using the Human Monocyte Isolation Kit II (Cat: 130–091–153; MACS, Miltenyi Biotech). The monocyte purity was >95% as confirmed by FACS analysis with an anti-CD14 antibody (BD Biosciences).
Freshly isolated human monocytes were differentiated into macrophages by incubation with either 50 ng/mL of human M-CSF (Cat: 14–8789) or G-CSF (Cat: 578602) (both from BioLegend) in RPMI medium (Cat: 61870–010; Gibco) supplemented with 1% heat-inactivated human AB serum (Cat: 34005100; Invitrogen), for up to 8 d. 20 ng/mL of recombinant human IL-4 (Cat: 204-IL; R&D Systems) was used as a control to induce M2-type macrophage differentiation and anti-human G-CSF (1 μg/mL, clone BVD13–3A5, Cat: 502101; BioLegend) or isotype control (rat IgG1 Cat: 400401) was used to neutralize G-CSF in MDA-MB-231 conditioned medium.

Hollmén M., Karaman S., Schwager S., Lisibach A., Christiansen A.J., Maksimow M., Varga Z., Jalkanen S, & Detmar M. (2015). G-CSF regulates macrophage phenotype and associates with poor overall survival in human triple-negative breast cancer. Oncoimmunology, 5(3), e1115177.

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Phenotypic Analysis of CD14+ Cells

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Purified CD14+ cells were stained with anti-CD14-antibody (BD Bioscience, San Jose, CA) and 7-Amino Actinomycin D (7-AAD) and incubated on ice for 30 minutes. Cells were analyzed using a FACS canto II flow cytometer (BD Bioscience, San Jose, CA) and data were analyzed using FlowJo v.X, (TreeStar, Inc. Ashland, OR).

Køllgaard T., Enevold C., Bendtzen K., Hansen P.R., Givskov M., Holmstrup P, & Nielsen C.H. (2017). Cholesterol crystals enhance TLR2- and TLR4-mediated pro-inflammatory cytokine responses of monocytes to the proatherogenic oral bacterium Porphyromonas gingivalis. PLoS ONE, 12(2), e0172773.

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CD11b and CD14 Expression Analysis

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For the CD14 or CD11b detection, cells (2×10⁵ cells/mL) were exposed to different concentrations of notopterol or ATRA (1 µM, as a positive control) for 96 hrs. Cells were harvested and washed twice with precold 1×PBS. Then, cells were incubated with direct fluorescein isothiocyanate-labeled anti-CD11b antibody (BD Biosciences) or anti-CD14 antibody (BD Biosciences) in the dark. After 30 mins, cells were washed twice with precold PBS and detected by flow cytometry (BD Biosciences).

Huang Q., Wang L., Ran Q., Wang J., Wang C., He H., Li L, & Qi H. (2019). Notopterol-induced apoptosis and differentiation in human acute myeloid leukemia HL-60 cells. Drug Design, Development and Therapy, 13, 1927-1940.

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Monocyte Stimulation and Epigenetic Modulation

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THP-1 cells (Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) and monocytes were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (Gibco), 2 mM L-Glutamine, 1 mM sodium pyruvate, and 10 mM HEPES at 37°C in a humidified atmosphere containing 5% CO2.
PBMCs were isolated using Ficoll-Paque PLUS (Cytiva). Monocytes (>98% purity) were sorted from the PBMCs with a BD FACSAriaTM III cell sorter (BD Biosciences). Monocytes were identified by anti-CD14 antibody (BD Biosciences).
For transient transfection, THP-1 cells (2 × 10⁵/well) were seeded in 24-well plate 24 h before transfection. SiRNAs (combination of four different EZH2 targeting siRNAs, 200 nM) were transfected with LipofectamineTM RNAiMAX (Invitrogen). Twenty-four hours later, the cells were stimulated with 1,000 U/ml of universal IFN-I, then collected for further analysis as shown in the figures. All siRNA sequences (GenePharma) are in Table S2.
For EZH2 inhibitor treatment, THP-1 cells were pretreated with 5 uM GSK126, 5 uM Dznep (Selleck Chemicals), or DMSO for 30 min, then 1,000 U/ml of universal IFN-I was added. Then, the cells were collected for further analysis as shown in the figures.

Wu L., Jiang X., Qi C., Zhang C., Qu B, & Shen N. (2021). EZH2 Inhibition Interferes With the Activation of Type I Interferon Signaling Pathway and Ameliorates Lupus Nephritis in NZB/NZW F1 Mice. Frontiers in Immunology, 12, 653989.

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Bone Marrow-Derived Dendritic Cell Culture

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The BMDC were prepared and cultured as described previously, with modifications.15 In brief, BM was flushed from tibias and femurs of C57BL/6J mice and after purification (depletion of T cells, B cells and MHC II⁺ cells), was cultured at 6 × 10⁵ cells/ml in bacteriological Petri dishes with complete RPMI‐1640 containing 10 ng/ml recombinant granulocyte–macrophage colony‐stimulating factor (GM‐CSF; R&D Systems, Minneapolis, MN). Fresh medium was added on days 2 and 4. On day 6 cells were harvested and depleted of contaminating granulocytes. The remaining cells were plated at 1 × 10⁶ cells/ml and after in vitro stimulation with LPS Escherichia coli 0111:B4 [standard purity grade LPS from Sigma (St Louis, MO), upLPS from Invivogen (San Diego, CA); 1 μg/ml] or M. tuberculosis extract [generated by sonication of non‐viable M. tuberculosis H37Ra purchased from Difco (BD, Franklin Lakes, NJ); 15 μg/ml] used for adoptive transfer experiments or analysis by flow cytometry, Western blotting or confocal microscopy. For some experiments BMDC were pre‐incubated with purified anti‐CD14 antibody (15 min, 10 μg/ml; BD Biosciences, San Jose, CA).

Klaska I.P., Muckersie E., Martin‐Granados C., Christofi M, & Forrester J.V. (2016). Lipopolysaccharide‐primed heterotolerant dendritic cells suppress experimental autoimmune uveoretinitis by multiple mechanisms. Immunology, 150(3), 364-377.

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Antibody-Dependent Cellular Phagocytosis Assay

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Human PBMCs were incubated with CFSE (ThermoFisher, San Diego, CA, USA) labeled SK-BR-3 cells at a 10:1 ratio in an ultra-low adherence 96-well plate. Antibody (samples or isotype control) was serially diluted and added to each well. Cells were subsequently incubated with anti-CD14 antibody (BD Biosciences, San Diego, CA, USA) to stain monocytes and propidium iodide (BD Biosciences, San Diego, CA, USA) to evaluate viability. After washing, 10,000 live CD14+ cells were acquired by FACS. The percent of live CD14+ cells that were positive for CFSE fluorescence was considered to be a direct measure of ADCP activity. Histograms representing the number of events and CFSE fluorescence intensity were plotted and overlaid. Histograms were generated using FlowJo^® data analysis software for visual comparison (Ashland, OR, USA).

Jassem S., Wang W., Sweet H., Manoukian R., Chow V., Kanakaraj P., Hutterer K.M., Kuhns S., Foltz I.N., Chen Q., Ferbas J, & McBride H.J. (2019). Functional and Nonclinical Similarity of ABP 980, a Biosimilar of Trastuzumab. Pharmaceutical Research, 36(12), 177.

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PBMC Isolation and LPS Stimulation

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Peripheral blood mononuclear cells (PBMCs) were isolated from blood using Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) in conjunction with Leucosep tubes (Greiner, Stonehouse, United Kingdom). The concentration of CD14-positive cells was determined using an anti-CD14 antibody (BD Biosciences, Oxfordshire, United Kingdom), and PBMCs were resuspended at 5 × 10⁶ CD14-positive cells/mL. For stimulation, cells were incubated for 1 hour at 37°C with Escherichia coli ultrapure lipopolysaccharide (LPS; Invivogen, San Diego, CA) at 1 μg/mL. To determine p38MAPK activation, PBMCs were fixed, permeated, and stained using antibodies specific for the phosphorylated (phospho)-p38MAPK (Thr180/Tyr182) isoform (Cell Signaling, Danvers, MA). An LPS stimulation of 1 μg/mL was used for 15 or 30 minutes. To measure the response, nonactivated MAPK phosphorylation levels were subtracted from LPS-activated levels. To determine sol-TNF production, PBMCs were stimulated for 4 hours at 37°C ± LPS (1 μg/mL). Supernatants were collected and sol-TNF levels measured by enzyme-linked immunosorbent assay (R&D Systems, Oxfordshire, United Kingdom). Patient interleukin (IL)-6 and sol-TNF levels were both measured by enzyme-linked immunosorbent assay of diluted heparinized plasma samples (Ebioscience, San Diego, CA).

O’Callaghan D.J., O’Dea K.P., Scott A.J., Takata M, & Gordon A.C. (2015). Monocyte Tumor Necrosis Factor-α–Converting Enzyme Catalytic Activity and Substrate Shedding in Sepsis and Noninfectious Systemic Inflammation*. Critical Care Medicine, 43(7), 1375-1385.

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Isolation of Monocytes and Lymphocytes from PBMC

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ACDA-treated blood from healthy donors was purchased from the Regional Center of Blood Donation and Blood Therapy in Krakow, Poland. Peripheral blood mononuclear cells (PBMC) were isolated by standard Pancoll human (Panbiotech, Aidenbach, Germany) density gradient centrifugation, washed and resuspended in RPMI 1640 medium (Corning), containing 10% heat-inactivated fetal bovine serum (EURx, Gdańsk, Poland). Monocytes were separated from PBMCs by counter-current centrifugal elutriation (JE-6B elutriation system equipped with a 5-mL Sanderson separation chamber; Beckmann-Coulter, Palo Alto, CA, USA), as described previously [24 (link)]. Cells were washed, resuspended in RPMI 1640 medium supplemented with 2 mM of L-glutamine, 5% heat-inactivated fetal bovine serum (EURx), and 25 µg/mL gentamycin (Sigma, St. Louis, MO, USA) (complete medium) and kept in an ice bath until used. Purity of isolated monocytes was checked by flow cytometry using anti-CD14 antibody (BD Biosciences, Pharmingen, San Diego, CA, USA) and did not drop below 90%. For some experiments, population of lymphocytes containing c.a. 80% of CD3-positive cells was isolated by counter-current centrifugal elutriation from PBMCs.

Gałuszka A., Stec M., Węglarczyk K., Kluczewska A., Siedlar M, & Baran J. (2020). Transition Metal Containing Particulate Matter Promotes Th1 and Th17 Inflammatory Response by Monocyte Activation in Organic and Inorganic Compounds Dependent Manner. International Journal of Environmental Research and Public Health, 17(4), 1227.

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Anti-CD14 Modulates Phagocytosis in LPS-Stimulated Kupffer Cells

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In some experiments, the phagocytosis assay was performed in the presence of anti-CD14 or the IgG control. KCs (isolated as described in 2.4) were incubated with 10 µg of the anti-CD14 antibody (BD Biosciences, Heidelberg, Germany) or the IgG control (BD Biosciences, Heidelberg, Germany) for 2 h. Subsequently, cells were stimulated with 10 ng/mL of LPS for 20 h. The phagocytosis assay was performed afterward, as described previously.

Liebold I., Meyer S., Heine M., Kuhl A., Witt J., Eissing L., Fischer A.W., Koop A.C., Kluwe J., zur Wiesch J.S., Wehmeyer M., Knippschild U., Scheja L., Heeren J., Bosurgi L, & Worthmann A. (2023). TREM2 Regulates the Removal of Apoptotic Cells and Inflammatory Processes during the Progression of NAFLD. Cells, 12(3), 341.

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Monocyte ROS and AML Superoxide Production

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ROS production by monocytes was assessed as previously described.¹⁰¹ Whole blood was subjected to red blood cell lysis and was then incubated with dihydrorhodamine 123 (DHR) in the presence or absence of phorbol 12-myristate 13-acetate (PMA, Sigma, 400 ng/mL) for 20 minutes at 37°C. Monocytes were labeled with an anti-CD14 antibody (#558121, BD, 1:50) and samples were analyzed on a Fortessa X20 flow cytometer. Superoxide production by AMLs was assessed with a superoxide anion assay kit (#CS1000, Merck) according to the manufacturer’s instructions. Briefly, 3 × 10⁴ AMLs were plated in 96-well plates 24 hours before the experiment. Luminescence, indicating superoxide production, was recorded with a Victor Nivo plate reader (PerkinElmer) immediately after stimulation with PMA (400 ng/mL) in the presence or absence of superoxide dismutase. Data were normalized against the values for superoxide dismutase-treated wells and are expressed in luminescence units.

Neehus A.L., Carey B., Landekic M., Panikulam P., Deutsch G., Ogishi M., Arango-Franco C.A., Philippot Q., Modaresi M., Mohammadzadeh I., Berndt M.C., Rinchai D., Le Voyer T., Rosain J., Momenilandi M., Martin-Fernandez M., Khan T., Bohlen J., Han J.E., Deslys A., Bernard M., Gajardo-Carrasco T., Soudée C., Le Floc’h C., Migaud M., Seeleuthner Y., Jang M.S., Nikolouli E., Seyedpour S., Begueret H., Emile J.F., Le Guen P., Tavazzi G., Julia Colombo C.N., Marzani F.C., Angelini M., Trespidi F., Ghirardello S., Alipour N., Molitor A., Carapito R., Mazloomrezaei M., Rokni-Zadeh H., Changi-Ashtiani M., Brouzes C., Vargas P., Borghesi A., Lachmann N., Bahram S., Crestani B., Pahari S., Schlesinger L.S., Marr N., Bugonovic D., Boisson-Dupuis S., Béziat V., Abel L., Borie R., Young L.R., Deterding R., Shahrooei M., Rezaei N., Parvaneh N., Craven D., Gros P., Malo D., Sepulveda F.E., Nogee L.M., Aladjidi N., Trapnell B.C., Casanova J.L, & Bustamante J. (2023). Human inherited CCR2 deficiency underlies progressive polycystic lung disease. Cell, 187(2), 390-408.e23.

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