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Anti human cd14 apc cy7

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Anti-human CD14-APC-Cy7 is a fluorochrome-conjugated monoclonal antibody that recognizes the CD14 cell surface antigen. CD14 is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein that serves as a co-receptor for the detection of bacterial lipopolysaccharide (LPS). The APC-Cy7 fluorochrome allows for multicolor flow cytometric analysis.

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Lab products found in correlation

Facscanto 2, by BD (2 mentions) 7 aad, by BD (2 mentions) Celltrace violet, by Thermo Fisher Scientific (2 mentions) Anti human cd34 apc, by BD (2 mentions) Anti human cd38 percp, by BioLegend (2 mentions) Celltrace violet cell proliferation kit, by Thermo Fisher Scientific (2 mentions) Anti human cd71 pe, by BioLegend (2 mentions) Anti human cd133 pe, by Miltenyi Biotec (2 mentions) Anti human cd11b pe cy7, by BioLegend (2 mentions) Facsverse flow cytometer, by BD (2 mentions) Apc anti human cd86, by BioLegend (1 mentions) Pe cy7 anti human hla dr, by BioLegend (1 mentions) Fitc anti human cd20, by BioLegend (1 mentions) Gentlemacs dissociator, by Miltenyi Biotec (1 mentions) Propidium iodide, by BioLegend (1 mentions) Pacific blue anti human cd3, by BioLegend (1 mentions) Fluorescein isothiocyanate antiphosphorylated ser139 h2ax clone 2f3, by BioLegend (1 mentions) Pe anti human cd8a, by BioLegend (1 mentions) Summit software v6, by Beckman Coulter (1 mentions) Nuclear factor fixation and permeabilization buffer set, by BioLegend (1 mentions)

Close spelling variants of this product

APC-cy7 anti-human CD14 (clone M5E2; Anti-CD14 APC-Cy7 APC anti-human CD14 CD14-APC-Cy7 Anti-CD14-APC CD14-APC APC-Cy7

8 protocols using anti human cd14 apc cy7

1

Tumor-infiltrating Immune Cell Profiling

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Tumor infiltrating CD45+ cells in two patients with NSCLC were analyzed using flow cytometry. There was one patient with squamous cell carcinoma and the other had adenocarcinoma of the lung. Tumor specimens from both patients were β-catenin negative by immunohistochemistry. CD8+ and CD11c+ cell infiltrations into tumor nests were positive in both cases. A 7-mm square of tissue was obtained from each patient's tumor at surgical resection. The tissue was dissociated into a single-cell suspension using a gentleMACS Dissociator (Miltenyi Biotec GmBH). Blood cells were separated and collected by CD45 MicroBeads and autoMACS separator (Miltenyi Biotec) and then stained with the following antibodies: PE anti-human CD11c (1:20; cat. no. 301605; BioLegend, Inc.), PE/Cy7 anti-human HLA-DR (1:20; cat. no. 307615; BioLegend, Inc.), PerCP/Cy5.5 anti-human CD163 (1:20; cat. no. 326511; BioLegend, Inc.), APC/Cy7 anti-human CD14 (1:20; cat. no. 301819; BioLegend, Inc.), APC anti-human CD86 (1:20; cat. no. 305411; BioLegend, Inc.), and FITC anti-human CD20 (1:20; cat. no. 130-113-373; Miltenyi Biotec GmBH). Samples were acquired with a FACSCanto II (BD Biosciences) and analysed using FlowJo software v10.0 (FlowJo LLC). The samples were gated on single cells, then the lymphocytes and monocytes were selected and analyzed.
Muto S., Ozaki Y., Yamaguchi H., Mine H., Takagi H., Watanabe M., Inoue T., Yamaura T., Fukuhara M., Okabe N., Matsumura Y., Hasegawa T., Osugi J., Hoshino M., Higuchi M., Shio Y., Nanamiya H., Imai J.I., Isogai T., Watanabe S, & Suzuki H. (2021). Tumor β-catenin expression is associated with immune evasion in non-small cell lung cancer with high tumor mutation burden. Oncology Letters, 21(3), 203.
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2

Immunophenotyping and Cell Cycle Analysis of PBMCs

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PBMCs were stained with Pacific Blue anti-human CD3, APC anti-human CD4, PE anti-human CD8a and APC/Cy7 anti-human CD14 (Bio Legend, San Diego, California, USA). The stained cells were then fixed and permeabilised using the Nuclear Factor Fixation and Permeabilization Buffer Set (BioLegend, San Diego, California, USA) as per the manufacturer's instructions. Intracellular staining was then performed using fluorescein isothiocyanate anti-phosphorylated (ser139) H2AX (clone 2F3; BioLegend). For the cell-cycle experiments, the PBMCs were additionally treated with 20 μg/mL RNase A (Sigma-Aldrich, St. Louis, Missouri, USA), and then stained with 40 μg/mL propidium iodide (BioLegend) as per manufacturer's instructions for 1 hour prior to analysis. Flow cytometry analysis was then performed using a MoFlow Astrios Flow Cytometer and Summit software V.6.2.3 (Beckman Coulter, Miami, Florida, USA). Phospho-H2AX levels are reported as median fluorescence intensity (MFI) values.
Namas R., Renauer P., Ognenovski M., Tsou P.S, & Sawalha A.H. (2016). Histone H2AX phosphorylation as a measure of DNA double-strand breaks and a marker of environmental stress and disease activity in lupus. Lupus Science & Medicine, 3(1), e000148.
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3

Identification of HIV Antibody-Producing Cells

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PBMC was purified by ficoll gradient from HIV infected patient, incubated with 1 μg/mlbiotinylated SF162gp140 trimer at 4°C for 30 min. The PBMC was then washed by PBS and stained with second antibodies:Pacific Blue™ anti-human CD19 (Biolegend, 302224), PE/Cy7 anti-human CD3(Biolegend, 300316), APC/Cy7 anti-human CD14 (Biolegend, 325620), APC anti-human IgG(Biolegend, 409306), 7-AAD(7-Aminoactinomycin D) (Invitrogen, 302232), FITC anti-human CD27(Biolegend, 302806), PerCP/Cy5.5 anti-human IgM (Biolegend, 314512), and R-Phycoerythrin Streptavidin (Jackson immunolab, 016-110-084). SF162gp140-trimerbinding memory B cells (CD19+, IgG+, SF162gp140+7AAD-IgM-CD14-CD3-) were sorted in 96 well PCR plate by FACS AriaIII(BD). The sorted cells were then lysedwith RNA directly reverse transcribed into cDNA according SuperScript™ III CellsDirectcDNA Synthesis System (Invitrogen, 18080-300) manual. Single cell derived cDNA was used for antibody variable gene amplification.
Yang Z., Liu X., Sun Z., Li J., Tan W., Yu W, & Zhang M. (2018). Identification of a HIV Gp41-Specific Human Monoclonal Antibody With Potent Antibody-Dependent Cellular Cytotoxicity. Frontiers in Immunology, 9, 2613.
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4

Phenotypic Analysis of CML CD34+ Cells

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CD34+ CML cells were stained with 1μM CellTrace Violet (CellTrace Violet Cell Proliferation Kit, Life Technologies) in PBS for 30 min at 37 °C. The reaction was quenched by adding cell culture medium containing 10% FBS. Cells were then washed and re-suspended in SFM supplemented with PGF cocktail and treated as indicated in figure legends. After 3 or 6 days, cells were stained with anti-human CD34-APC (BD Biosciences), anti-human CD38-PerCP (BioLegend) and anti-human CD133-PE (Miltenyi Biotec). For detection of differentiation markers, cells were stained with anti-human CD71-PE, anti-human CD11b-PE-Cy7 and anti-human CD14-APC-Cy7 (all from BioLegend) followed by flow cytometry analysis (FACSVerse™ Flow Cytometer, BD Biosciences). Data analysis was performed using FlowJo 7.6.5 software.
Baquero P., Dawson A., Mukhopadhyay A., Kuntz E.M., Mitchell R., Olivares O., Ianniciello A., Scott M.T., Dunn K., Nicastri M.C., Winkler J.D., Michie A.M., Ryan K.M., Halsey C., Gottlieb E., Keaney E.P., Murphy L.O., Amaravadi R.K., Holyoake T.L, & Helgason G.V. (2018). Targeting quiescent leukemic stem cells using second generation autophagy inhibitors. Leukemia, 33(4), 981-994.
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5

Characterization of CD34+ CML Cells

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CD34+ CML cells were stained with 1 μM CellTrace Violet (CellTrace Violet Cell Proliferation Kit, Life Technologies) in PBS for 30 min at 37 °C. The reaction was quenched by adding cell culture medium containing 10% FBS. Cells were then washed and re-suspended in SFM supplemented with PGF cocktail and treated as indicated in figure legends. After 3 or 6 days, cells were stained with anti-human CD34-APC (BD Biosciences), anti-human CD38-PerCP (BioLegend) and anti-human CD133-PE (Miltenyi Biotec). For detection of differentiation markers, cells were stained with anti-human CD71-PE, anti-human CD11b-PE-Cy7 and anti-human CD14-APC-Cy7 (all from BioLegend) followed by flow cytometry analysis (FACSVerseTM Flow Cytometer, BD Biosciences). Data analysis was performed using FlowJo 7.6.5 software.
Baquero P., Dawson A., Mukhopadhyay A., Kuntz E.M., Mitchell R., Olivares O., Ianniciello A., Scott M.T., Dunn K., Nicastri M.C., Winkler J.D., Michie A.M., Ryan K.M., Halsey C., Gottlieb E., Keaney E.P., Murphy L.O., Amaravadi R.K., Holyoake T.L, & Helgason G.V. (2018). Targeting quiescent leukemic stem cells using second generation autophagy inhibitors. Leukemia, 33(4), 981-994.
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6

Calcium Signaling in Monocyte-Derived Macrophages

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MDMs were re-suspended at 1E7 cells per mL in Hanks Buffered Salt Solution with Ca2+ and MgCl2 (HBSS, HyClone, SH 30268.02), 1% FBS (HyClone, SH30071.03, Thermo Scientific), and 4mM Probenecid (Invitrogen, P36400). MDMs were treated with 1:100 anti-human CD-16 Brilliant Violet 605TM (BioLegend, 302040) and 1:100 anti-human CD-14-APC-Cy7 (BioLegend, 325620) to identify macrophage populations. The MDMs were incubated at 37°C for 30 min with 4µg/mL fluorescent dye Fluo-3 AM (Life Technologies, F1242), washed twice and re-suspended in cell loading HBSS medium at 1E7 cells/mL. MDMs were stimulated with 1µg/mL Ionomycin (Sigma-Aldrich, 124222), 1µM Platelet Activating Factor (PAF, Sigma-Aldrich, P4904), bacteria (MOI 10) or PMA (Calbiochem, 524400). The intensity of intracellular Ca2+ in individual cells was assessed by flow cytometer (LSR II, BD) measuring the fluorescence emission of Fluo-3 in the FL-1 channel over 200s. Data were analyzed using FlowJo software (Tree Star. San Carlos, California).
Assani K., Shrestha C.L., Robledo-Avila F., Rajaram M.V., Partida-Sanchez S., Schlesinger L.S, & Kopp B.T. (2017). Human Cystic Fibrosis Macrophages Have Defective Calcium-Dependent PKC Activation of the NADPH Oxidase, an Effect Augmented by Burkholderia cenocepacia. Journal of immunology (Baltimore, Md. : 1950), 198(5), 1985-1994.
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7

Immunoglobulin and Nanoparticle Characterization

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Privigen® human immunoglobulin from CSL Behring (King of Prussia, PA, USA) was used for flow cytometry. Anti-human-CD14-APC-Cy7 and anti-human-CD19-PE-Cy7 were from BioLegend (San Diego, CA, USA), anti-human-CD3-Pacific-blue and Annexin V/7AAD apoptosis kits were from BD Biosciences (San Jose, CA, USA), and anti-human-CD15-APC was from Miltenyi Biotech GmbH (Bergisch Gladbach, Germany). Lectin from Phaseolus vulgaris (PHA-L) and lipopolysaccharide (LPS) from Sigma-Aldrich Co. (St Louis, MO, USA) were used for immune cell stimulation. The commercially available MRI contrast agent ferumoxsil (Lumirem®; Guerbet, Villepinte, France) was used as a negative control for the final comparative analysis of different NP preparations.
Strehl C., Maurizi L., Gaber T., Hoff P., Broschard T., Poole A.R., Hofmann H, & Buttgereit F. (2016). Modification of the surface of superparamagnetic iron oxide nanoparticles to enable their safe application in humans. International Journal of Nanomedicine, 11, 5883-5896.
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8

Measurement of Cellular Oxidative Stress

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To measure cellular oxidative stress, iROS were detected using 5 (and 6) - chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA; Invitrogen GmbH, Karlsruhe, Germany). Cells were incubated 30 min in PBS (DRFZ, Berlin, Germany) with 5 µM CM-H2DCFDA, washed with glucose-free RPMI-1640 and incubated 2 hours in glucose-free RPMI-1640 and 10% (v/v) dialyzed human AB serum, with or without 100 ng/mL LPS and with 1% DMSO, MYX1 or MYX2. After incubation, cells were washed with PBS and subsequently stained with anti-human CD14-APC-Cy7 (Biolegend, San Diego, USA). To exclude dead and apoptotic cells, cells were stained with Annexin V-PE (Biolegend, San Diego, USA) and 7-AAD (BD Biosciences, San Jose, USA) (gating strategy provided in Figure S3, reagent concentration provided in Tables 1, 2). Data were acquired using a BD FACSCanto™ II (BD Biosciences, San Jose, USA) and processed by FlowJo v7.6.5 (BD Biosciences, San Jose, USA).
Krauss P.L., Pfeiffenberger M., Damerau A., Buttgereit T., Chen Y., Gaber T, & Buttgereit F. (2021). Production of IL-6 and Phagocytosis Are the Most Resilient Immune Functions in Metabolically Compromised Human Monocytes. Frontiers in Immunology, 12, 730672.
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