Pe conjugated anti cd14

Manufactured by BD

Sourced in United States

PE-conjugated anti-CD14 is a monoclonal antibody that binds to the CD14 receptor. CD14 is a cell surface glycoprotein that serves as a co-receptor for the detection of lipopolysaccharides. The PE (Phycoerythrin) fluorescent label allows for the detection and analysis of CD14-expressing cells using flow cytometry.

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Lab products found in correlation

Apc conjugated anti cd4, by BD (2 mentions) Fitc conjugated anti cd45, by BD (2 mentions) Fitc conjugated anti cd16, by BD (2 mentions) Fc500 flow cytometer, by Beckman Coulter (2 mentions) Acutase, by Merck Group (1 mentions) Bapta am, by Merck Group (1 mentions) Complete protease inhibitor tablet, by Roche (1 mentions) Hrp conjugated goat anti rabbit ig, by Agilent Technologies (1 mentions) Ovalbumin (ova), by Merck Group (1 mentions) Heparin, by Leo pharma (1 mentions) Phosphate buffered saline (pbs), by Boster Bio (1 mentions) Fitc conjugated anti cd105, by BD (1 mentions) Influx cell sorter, by BD (1 mentions) Pe conjugated anti cd34, by BD (1 mentions) Apc conjugated anti cd45, by BD (1 mentions) Fitc conjugated anti cd271, by BioLegend (1 mentions) Fc receptor blocking solution, by Miltenyi Biotec (1 mentions) Fitc conjugated anti cd31, by BD (1 mentions) Lsr 2, by BD (1 mentions) Pe conjugated anti cd86, by BD (1 mentions)

Spelling variants of this product

CD14-PE (78 citations) Anti-CD14-PE (40 citations) PE-conjugated CD14 (3 citations)

9 protocols using pe conjugated anti cd14

Recombinant SOD3 Protein Production and Antibody Development

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Human serum, M-CSF, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), acutase, and ovalbumin was from Sigma. Heparin (5000 U/ml) was obtained from Leo Pharma, Denmark and complete protease inhibitor tablets was from Roche.
Proteins: Recombinant wild-type SOD3 and SOD3 containing a C-terminal c-Myc tag (SOD3-cMyc) was produced as previously described [42 (link)]. For the generation of the SOD3-cMyc expression plasmid, we used the forward primer (5′-TATACAGCTAGCATGCTGGCGCTACTGTGTTCC-3′) and a reverse primer encompassing the cMyc tag (underlined) (5′-TATGAATTCTCACAGATCCTCTTCTGAGATGAGTTTTTGTTCGGCGGCCTTGCACTCGCTCTC-3′) and cloned the product into the pIRES vector. The sequence of the obtained plasmid was verified by sequencing. mAb 5G8D4 and 7F6D9 anti-human SOD3 (GenScript) and rabbit anti SOD3 antisera (Davids Biotechnologie) were developed using recombinant human SOD3 as antigen and antibodies recovered by using protein G-Sepharose. Recombinant human and murine IFNγ was obtained from Invitrogen. ovalbumin was from Sigma. Human receptor-associated protein (RAP) was obtained from ENZO life sciences (BML-SE552). HRP-conjugated goat anti-rabbit Ig was from DAKO. Flow cytometric analyses were performed using PE-conjugated anti-CD14 (B&D Biosciences), APC-conjugated anti-CD4 (B&D Biosciences) or APC-conjugated mAb5G8D4 anti-SOD3.

Hu L., Zachariae E.D., Larsen U.G., Vilhardt F, & Petersen S.V. (2019). The dynamic uptake and release of SOD3 from intracellular stores in macrophages modulates the inflammatory response. Redox Biology, 26, 101268.

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Flow Cytometry Analysis of MSCs

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MSCs were digested and washed with phosphate-buffered saline (PBS, Boster, Wuhan, China). After incubation with specific antibodies, MSCs were washed again and resuspended in PBS. Flow cytometry was performed using a BD Biosciences Influx Cell Sorter (San Jose, CA, USA). Human phycoerythrin- (PE-) conjugated anti-CD29, fluorescein isothiocyanate- (FITC-) conjugated anti-CD73, FITC-conjugated anti-CD105, FITC-conjugated anti-CD45, PE-conjugated anti-CD14, and PE-conjugated anti-HLA-DR antibodies were used for these experiments (all from BD Biosciences, San Diego, CA, USA).

Wu X., Ma M., Wang P., Xie Z., Wang S., Su H., Deng W., Feng P., Su C., Yang J., Li J., Tang S., Wu Y, & Shen H. (2019). α2-HS Glycoprotein in Plasma Extracellular Vesicles Inhibits the Osteogenic Differentiation of Human Mesenchymal Stromal Cells In Vitro. Stem Cells International, 2019, 7246479.

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Comprehensive Immunophenotyping of Isolated Cells

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Cell concentration was counted using a cell chamber. For the quantification of cell types among the isolated cells, the cells were immunophenotyped using a panel of fluorescence-conjugated antibodies. The cells were incubated with Fc- receptor blocking solution (Miltenyi) to block unspecific binding of antibodies, followed by incubation with specific antibodies according to manufacturer recommendations. Panel of used fluorescence-conjugated antibodies in the study as followed: PE-conjugated-anti-CD44 (Beckman Coulter, cat.no: A32537), PE-conjugated-anti-CD90 (Beckman Coulter, cat.no: IM3600U), PE-conjugated-anti-CD105 (Beckman Coulter, cat.no: A07414), FITC-conjugated-anti-CD271 (Biolegend, cat.no: 345104), FITC-conjugated-anti-CD31 (BD Pharmingen, cat.n. 555445), PE-conjugated-anti-CD34 (BD Pharmingen, cat.no: 555822), PE-conjugated-anti CD14 (BD Pharmingen, cat.no: 555398) and APC-conjugated-anti-CD45 (BD Pharmingen, cat.n. 555485), FITC-LEPR (R&D systems, cat.n. FAB867F), PE-SOX2 (R&D systems, cat.n. IC2018P), PE-CD49a (BD Pharmingen, cat.n. 559596). Following incubation for 30 min in the dark at 4 °C, cells were washed and analysed by BD LSR II (BD Biosciences). The flow cytometry analysis was performed by Kaluza 1.1 analysis software. Flow cytometric gating was defined based on relevant isotype controls.

Tencerova M., Lundby L., Buntzen S., Norderval S., Hougaard H.T., Pedersen B.G, & Kassem M. (2021). Molecular differences of adipose-derived mesenchymal stem cells between non-responders and responders in treatment of transphincteric perianal fistulas. Stem Cell Research & Therapy, 12, 586.

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Caspase-1 Activity in CAPS Patient PBMCs

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PBMCs were obtained from patients CAPS (n = 5; three males; median age, 3 yr; range, 2.5–7.3 yr; 1 with chronic infantile neurological and cutaneous (CINCA) syndrome; and 4 with Muckle Wells Syndrome; CAPS disease activity score median 1/20, range 0–4/20; all treated with canakinumab); and from four healthy adult controls.
PBMCs were seeded in a 96-well plate at a density of 1.6 × 10⁵ cells/well (8.0 × 10⁵ cells/ml). Relevant wells were primed with 100 ng/ml LPS for 4 h, and, if required, then stimulated with 5 mM ATP for 30 min. Additional wells were treated with 2 µM staurosporine for 4 h as positive control. Caspase-1 activity was measured using FLICA (ImmunoChemistry Technologies), a cell-permeable fluorescent probe (FAM-YVAD-FMK) that binds active caspase-1. Cells were incubated for 1 h with FLICA at 37°C and stained with PE-conjugated anti-CD14 (BD) to identify monocytes. The gating strategy consisted of including live CD14⁺ cells, which were subsequently analyzed for the frequency of FLICA⁺ cells.

Standing A.S., Malinova D., Hong Y., Record J., Moulding D., Blundell M.P., Nowak K., Jones H., Omoyinmi E., Gilmour K.C., Medlar A., Stanescu H., Kleta R., Anderson G., Nanthapisal S., Gomes S.M., Klein N., Eleftheriou D., Thrasher A.J, & Brogan P.A. (2017). Autoinflammatory periodic fever, immunodeficiency, and thrombocytopenia (PFIT) caused by mutation in actin-regulatory gene WDR1. The Journal of Experimental Medicine, 214(1), 59-71.

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Flow Cytometric Analysis of Cell Surface Markers

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Cell surface markers were analysed by immunostained with PE-conjugated anti-CD14 and FITC-conjugated anti-CD16 (BD Biosciences, San Jose, CA, USA). Cells were suspended with FACs buffer solution and directly stained with fluorescence-conjugated antibody. The reaction was incubated at 4 °C for 1 h in the dark and the cells were washed twice with PBS. Cell pellet was resuspended with staining buffer and analyzed by FC 500 Flow Cytometer (Beckman Coulter, Inc., Indianapolis, IN, USA). FITC-conjugated mouse IgG1,κ isotype control and PE-conjugated mouse IgG1,κ isotype control (BioLegend, Inc., San Diego, CA, USA) were used as a negative control.

Luetragoon T., Pankla Sranujit R., Noysang C., Thongsri Y., Potup P., Suphrom N., Nuengchamnong N, & Usuwanthim K. (2020). Bioactive Compounds in Moringa oleifera Lam. Leaves Inhibit the Pro-Inflammatory Mediators in Lipopolysaccharide-Induced Human Monocyte-Derived Macrophages. Molecules, 25(1), 191.

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Characterization of Monocyte Phenotype

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To study surface molecules expression, cells were washed with PBS, removed with a cell scraper, and labeled with human FITC-conjugated anti-CD14 (BioLegend, USA) or PE-conjugated anti-CD14 (BD Pharmingen, USA) and PE-conjugated anti-HLA-DR (BD Pharmingen, USA), PE-conjugated anti-CD86 (BD Pharmingen, USA) or FITC-conjugated anti-CD80 (BD Pharmingen, USA), and FITC-conjugated anti-CD40 (BD Pharmingen, USA) or PE-conjugated anti-CD69 (BD Pharmingen, USA) for 30 minutes at 4°C.
Cells were then washed twice with PBS + FCS 5% (200 ×g, 7 minutes) and resuspended in the same solution in the cold. Fluorescence measurements were performed using a FACScan or a FACSCalibur flow cytometer (Beckton, Dickinson and Company, USA). Fluorescence signals for no less than 10,000 cells were recorded in the monocyte gate, which was determined according to cell size and complexity parameters. Using these parameters, approximately 98% of the cells in this gate were CD14⁺. Data analyses were performed using Summit v4.3 software (Dako, USA).

Teixeira M.P, & Rumjanek V.M. (2014). Ouabain Affects the Expression of Activation Markers, Cytokine Production, and Endocytosis of Human Monocytes. Mediators of Inflammation, 2014, 760368.

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Macrophage Cell Surface Marker Analysis

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The expressions of macrophages cell surface markers were evaluated by staining with PE-conjugated anti-CD14 and FITC-conjugated anti-CD16 (BD Biosciences, San Jose, CA, USA). Harvested cells were centrifuged and resuspended in FACS buffer containing fluorescence-conjugated antibodies at 4 °C for 1 h. After incubation, the cells were washed three times with phosphate-buffered saline (PBS) and analyzed using an FC 500 Flow Cytometer (Beckman Coulter, Inc., Indianapolis, IN, USA). FITC-conjugated mouse IgG1, κ isotype control and PE-conjugated mouse IgG1, κ isotype control (BioLegend, Inc., San Diego, CA, USA) were used as negative control.

Buakaew W., Pankla Sranujit R., Noysang C., Thongsri Y., Potup P., Nuengchamnong N., Suphrom N, & Usuwanthim K. (2021). Phytochemical Constituents of Citrus hystrix DC. Leaves Attenuate Inflammation via NF-κB Signaling and NLRP3 Inflammasome Activity in Macrophages. Biomolecules, 11(1), 105.

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Characterizing Immune Cell Phenotypes

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Monocytes were stained using fluorescein isothiocyanate (FITC)conjugated anti-CD45 (BD Biosciences, San Jose, CA, USA), phycoerythrin (PE)-conjugated anti-CD14 (BD Biosciences), peridininÀchloro-phyllÀproteinÀconjugated anti-CD3 (BD Biosciences) and BD Via Probe Cell Viability Solution (7-aminoactinomycin D [7-AAD]; BD Pharmingen, San Diego, CA, USA). PMNs were stained using FITC-conjugated anti-CD45, allophycocyanin (APC)-conjugated anti-CD10 (BD Biosciences) and BD Via Probe Cell Viability Solution (7-AAD). In vitroÀexpanded T lymphocytes were stained using FITC-conjugated anti-CD45, peridininÀchlorophyllÀproteinÀconjugated anti-CD3, APC-conjugated anti-CD4 (BD Biosciences), PE-conjugated anti-CD8 (BD Biosciences) and BD Via Probe Cell Viability Solution (7-AAD). To assess TRAIL receptor expression, monocytes, PMNs and T lymphocytes were tested for PE-conjugated anti-DR4 (BioLegend, San Diego, CA, USA), APC-conjugated anti-DR5 (BioLegend), PE-conjugated anti-DcR1 (BioLegend) and APC-conjugated anti-DcR2 (R&D Systems, Minneapolis, MN, USA). Isotype control antibodies were used for all cell types and antigens analyzed. All samples were acquired using the BD FACSAria III (BD, Franklin Lakes, NJ, USA) flow cytometer and analyzed using the BD FACSDiva software.

Casari G., Dall'Ora M., Melandri A., Masciale V., Chiavelli C., Prapa M., Neri G., Spano M.C., Murgia A., D'Esposito A., Baschieri M.C., Ceccherelli G.B., Dominici M, & Grisendi G. (2023). Impact of soluble tumor necrosis factor-related apoptosis-inducing ligand released by engineered adipose mesenchymal stromal cells on white blood cells. Cytotherapy, 25(6).

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Isolation and Stimulation of Blood Cells

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Peripheral blood mononuclear cells from healthy volunteers were isolated by Ficoll densitygradient centrifugation. B cells from blood donors were isolated using the MS columns and the CD19 Microbeads kit (Miltenyi Biotec, Bergisch Gladbach, Germany) following manufacturer's protocols. Monocytes were isolated by positive selection using MS columns and the CD14 Microbeads kit (Miltenyi Biotec, Bergisch Gladbach, Germany). To assess the cell purity by flow cytometry analysis, B cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD20 (Miltenyi Biotec, Bergisch Gladbach, Germany), and monocytes were labeled with phycoerythrin (PE)-conjugated anti-CD14 (BD Biosciences, Le Pont de Claix, France). Isolated cells had an average purity of 90% (± 8%) as determined by flow cytometry analysis.
PBMCs were cultured in 24-well plates at 2 x 10 6 cells/ml in RPMI with 10% FBS at 37°C, 5% CO 2 . Isolated CD14 + cells were cultured at 2.5 x 10 5 in 24-well plates in DMEM with 5% FBS at 37°C, 5% CO 2 . Isolated CD19 + cells were cultured at 10 5 cells in 96-well plates in RPMI 10% FBS at 37°C, 5% CO 2 . Cultured cells were incubated in the presence or absence of 50 UI/ml of human recombinant IFN-γ (ImmunoTools, Friesoythe, Germany). After 6 hours, CD14 + and CD19 + cells were harvested for RNA extraction, whilst after 24 hours, PBMCs were collected for FACS analysis.

Miatello J., Lukaszewicz A., Faivre V., Hua S., Martinet K.Z., Bourgeois C., Quintana‐Murci L., Payen D., Boniotto M., & Tissières P. (2021). CIITA G-286A promoter polymorphism impairs monocytes HLA-DR expression in septic shock and is rescued by interferon-γ.

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