Pe anti human cd14

Manufactured by BioLegend

Sourced in United States

PE anti-human CD14 is a fluorescent-conjugated antibody that binds to the CD14 antigen on the surface of human cells. CD14 is a co-receptor for the detection of bacterial lipopolysaccharide (LPS) and is expressed on monocytes, macrophages, and neutrophils. This product can be used for the identification and enumeration of CD14-positive cells using flow cytometry.

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CD14-PE (26 citations) Anti-CD14-PE (9 citations)

9 protocols using pe anti human cd14

Comprehensive Immune Cell Profiling

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CBC and T-cell analyses were performed by Karolinska University Laboratory, Stockholm, Sweden, using Sysmex XN-9000 for CBC processing. T-cell analysis for CD4+ and CD8+ expression was performed using an Aquios CL (Beckman coulter) which utilizes a direct volumetric single‐platform method with incorporated sample preparation with a monoclonal antibody mixture (anti‐CD45‐FITC [clone B3821F4A], anti‐CD4‐RDI [clone SFCI12T4D11], anti CD8‐ECD [SFCI21thyD3], anti‐CD3‐PC5 [clone UCHT1]) Beckman Coulter.
For PBMC isolation, whole blood was sampled using BD Vacutainer^® CPT™ Mononuclear Cell Preparation Tubes and processed within 3 hours of collection. PBMCs were then isolated according to the standard procedure (centrifuged at 1500 g for 20 min at room temperature) and washed with cold PBS (440 g for 10 min at 4°C). Single cell suspensions were plated in 96-well V-bottomed plates and stained for 20 min at 4°C. The cells were incubated with Alexa Fluor647 anti-human CX3CR1 (clone: 2A9-1, BioLegend), PerCP/Cy5.5 anti-human CD192 (CCR2) (clone: K036C2, BioLegend), APC/Cy7 anti-human CD68 (clone: Y1/82A, BioLegend), PE/Cy7 anti-human CD11b (clone: ICRF44, BioLegend), Alexa Fluor488 anti-human CD16 (clone: 3G8, BioLegend) and PE anti-human CD14 (clone: 63D3, BioLegend). Cells were acquired using a Gallios flow cytometer (Beckman Coulter) and analyzed using Kaluza software (Beckman Coulter).

Hildenborg M., Kåhlin J., Granath F., Schening A., Granström A., Ebberyd A., Klevenvall L., Zetterberg H., Han J., Schlegel T.T., Harris R., Harris H.E, & Eriksson L.I. (2022). The Neuroimmune Response to Surgery – An Exploratory Study of Trauma-Induced Changes in Innate Immunity and Heart Rate Variability. Frontiers in Immunology, 13, 911744.

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Immune Cell Phenotyping Protocol

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Blood samples were centrifuged and washed four times with PBS. The buffy coat was collected from the blood with a pipette. Virus infection and fixation followed the procedure described above. The cells were stained with DAPI, PerCP-Cy5.5 anti-human CD45 (304027; BioLegend, San Diego, CA, USA); and one of the following antibodies: PE anti-human CD2 (309207), PE anti-human CD13 (301703), PE anti-human CD14 (301805), PE anti-human CD15 (301905), PE anti-human CD19 (302207), PE anti-human CD45 (304008) (all from BioLegend), and PE anti-human CD203c (130092243; Miltenyi Biotec Inc., San Diego, CA, USA).

YABUSAKI M., SATO J., KOHYAMA A., KOJIMA T., NOBUOKA D., YOSHIKAWA T., SAWADA Y., MURAKAMI K., GOHDA K., OKEGAWA T., NAKAMURA M., TAKAMATSU K., ITO M., KANEKO K, & NAKATSURA T. (2014). Detection and preliminary evaluation of circulating tumor cells in the peripheral blood of patients with eight types of cancer using a telomerase-specific adenovirus. Oncology Reports, 32(5), 1772-1778.

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Immunophenotyping of Blood Cell Subsets

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The blood samples were centrifuged to collect the plasma. White blood cells were obtained after red blood cells were lysed and centrifuged at 1000 rpm. After incubating with Fc receptor blocking solution (Human TruStain FcX™, Biolegend, California, USA) for 30 min., the cells were further incubated with FITC anti-human CD19, APC anti-human CD4 and PE anti-human CD14 (Biolegend, California, USA) for 30 min. Flow cytometry analysis was done in FACS buffer containing PBS plus 2% FBS using BD LSRFortessa instrument. FlowJo v10.2 software (Treestar) was used for data analysis. Cells were sorted using BD FACSAria.

Fang Q., Li T., Chen P., Wu Y., Wang T., Mo L., Ou J, & Nandakumar K.S. (2021). Comparative Analysis on Abnormal Methylome of Differentially Expressed Genes and Disease Pathways in the Immune Cells of RA and SLE. Frontiers in Immunology, 12, 668007.

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Characterization of Circulating Tissue Factor-Positive Microparticles

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Circulating MPs were isolated using differential centrifugation, as previously described^{15 (link))}. Briefly, platelets were removed by two subsequent centrifugations at 2,500 g for 15 minutes at room temperature. Then the supernatant was centrifuged at 18,000 g for 60 minutes to pellet the MPs. The pellets were resuspended by annexin V binding buffer and then incubated with FITC-anti-annexin V (BD Pharmingen) and APC-antihuman TF (BioLegend) for 30 minutes at 4°C in the dark. Then 0.8 µm and 3 µm beads were used for gating and counting control, respectively. Circulating TF-positive MPs were detected as annexin V⁺/TF⁺ particles by a FACS Calibur cytometer (BD Biosciences)^{16 (link))}. To investigate the cellular origin of circulating TF-positive MPs, samples were incubated with PE-anti-Human CD41 or PE-anti-Human CD14 (BioLegend). Analysis was performed using FlowJo (Tree Star Inc.) software.

Wang G.H., Lu J., Ma K.L., Zhang Y., Hu Z.B., Chen P.P., Lu C.C., Zhang X.L, & Liu B.C. (2019). The Release of Monocyte-Derived Tissue Factor-Positive Microparticles Contributes to a Hypercoagulable State in Idiopathic Membranous Nephropathy. Journal of Atherosclerosis and Thrombosis, 26(6), 538-546.

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Differentiation of Dendritic Cells from PBMCs

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PBMCs were collected from healthy volunteers and cultured in AIM-V medium (A3021002, Gibco) at 37°C and 5% CO_2. Two hours later, cell flasks were gently shaken to suspend and remove the unattached and semi-attached cells, which were cultured to induce immature DCs (iDCs) in AIM-V medium supplemented with 800U/ml GM-CSF (215-GMP-050, R&D Systems) and 500U/ml IL-4 (204-GMP-050, R&D Systems) for 6 days. The supernatant of iDCs was mixed with fresh medium in a ratio of 1:1 to induce mature DCs (mDCs) for 16–18 h with 800U/ml GM-CSF, 500U/ml IL-4, 160ng/ml IL-6 (206-GMP-050, R&D Systems), 5ng/ml IL-1β (201-GMP-100, R&D Systems), 5ng/ml TNFα (210-GMP-100, R&D Systems), and 1μg/ml PGE2 (2296/10, Tocris). The iDCs and mDCs were identified by flow cytometry using PE-anti-human CD80 (305208, BioLegend), PE-anti-human CD83 (305308, BioLegend), PE-anti-human CD86 (305406, BioLegend), PE-anti-human CD14 (367104, BioLegend), PE-anti-human CD11c (301606, BioLegend), PE-anti-human HLA-DR (307606, BioLegend), PE-anti-human HLA-ABC (311406, BioLegend), and PE-anti-human CCR7 (353204, BioLegend) antibodies.

Chen R., Huang M., Yang X., Chen X.H., Shi M.Y., Li Z.F., Chen Z.N, & Wang K. (2021). CALR-TLR4 Complex Inhibits Non-Small Cell Lung Cancer Progression by Regulating the Migration and Maturation of Dendritic Cells. Frontiers in Oncology, 11, 743050.

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Monocyte Differentiation Assay Protocol

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10 mM PMA (Sigma) and 100 mM ATRA (Sigma) were dissolved in DMSO and stored at −20°C. Compounds were diluted to 3 nM (PMA) and 3 μM (ATRA) in respective media and cells were suspended at 100,000 cells/ml in 6-well non-tissue culture plates. Cell differentiation was detected by flow cytometry, staining with PE anti-human CD14 (BioLegend) and APC anti-mouse/human CD11b (BioLegend) from cells sampled from culture every 24 hours. All sample data was collected on BD FACSCanto instrument and analyzed using FlowJo software.

Lewis A.H., Bridges C.S., Punia V.S., Cooper A.F., Puppi M, & Lacorazza H.D. (2021). Krüppel-like factor 4 promotes survival and expansion in acute myeloid leukemia cells. Oncotarget, 12(4), 255-267.

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Multiparameter Flow Cytometry of Hematopoietic Cells

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BM, spleen (SPL), and peripheral blood (PB) were obtained from mice. Single-cell suspensions were prepared from each tissue following standard procedures. Erythrocytes were hemolyzed using BD Pharm Lyse buffer (BD Biosciences). Leukocytes were stained with fluorochrome-conjugated human antibody/mouse antibody for 20 min at room temperature in FACS buffer. After washing the labeled cells in 1x PBS, the cells were re-suspended in FACS buffer. Fluorescent signals were detected with a FACSVerse^TM flow cytometer, and data were analyzed using FlowJo software (BD Biosciences). The following human antibodies were used to measure human engrafted hematopoietic cells and mouse hematopoietic cells: anti-human CD45-APC and anti-human CD66b-FITC were purchased from Beckman Coulter. Anti-mouse CD45-FITC, anti-human CD33-FITC, anti-human CD14-PE, anti-human CD11b-PE, anti-human CD41-PE, anti-human CD235a-FITC, anti-human CD3-FITC, anti-human CD19-APC were purchased from BioLegend.

Katahira Y., Higuchi H., Matsushita H., Yahata T., Yamamoto Y., Koike R., Ando K., Sato K., Imadome K.I, & Kotani A. (2019). Increased Granulopoiesis in the Bone Marrow following Epstein-Barr Virus Infection. Scientific Reports, 9, 13445.

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Monocyte-to-M1 Macrophage Differentiation

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To evaluate the degree of differentiation of monocytes to M1 macrophages after 7 days of stimulation with GM-CSF, the macrophages and monocytes (unstimulated cells) were removed with scraper, washed with FACS buffer (1X PBS with 3% FBS) and surface marked with anti-Human CD14- PE (Clone M5E2, Biolegend, San Diego, CA), anti-Human CD11b-APC (Clone ICRF44, Biolegend, Cat# 301310), Anti-Human CD1a-BV711 (Clone HI149, Biolegend, Cat# 300139) and Anti-Human CD163-APC-Cy7 (Clone GHI/61, Biolegend, Cat# 333621) at 4 °C for 30 min. The cells were checked for CD14 purity by flow-cytometry in BD LSRFortessa^TM X-20 (BD Biosciences).

Konduru N.V., Velasco-Alzate K., Adduri S., Zagorovsky K., Diaz-Diestra D., Fisol F., Sanches M., Ndetan H., Brain J.D, & Molina R.M. (2021). Pulmonary fate and consequences of transferrin-functionalized gold nanoparticles. Nanotheranostics, 5(3), 309-320.

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Phagocytosis Assay for Therapeutic Antibodies

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CD14⁺ cells were isolated from human PBMCs using anti-human biotin CD14⁺ antibody (BioLegend; Cat. 325624) and cultured in macrophage differentiation media (RPMI1640 + 20% FBS + 1% P/S + 20 ng/mL M-CSF) for 6 days and 30,000 cells seeded overnight in a new plate. On day 7, 60,000 RAJI-GFP cells (target cells) were preincubated with STI-6643 or rituximab for 30 minutes at 37°C followed by addition to the plate containing the macrophages and incubated for 1 hour. Macrophages were labelled with anti-human CD11b PE (BioLegend; Cat. 982606) for 15 minutes prior the end of the incubation. Phagocytosis of target cells were measured as percent CD11b⁺GFP+ macrophages on Attune flow cytometer.
Alternately, Cell Proliferation Dye eFluor™ 670 (eBioscience; Cat. 65-0840-85) was used to label MDA-MB-231-Fluc target cells and RAJI-GFP were used without prior labelling. MDA-MB-231 cells were incubated for 2 h at 37°C prior to adding anti-CD47 (STI-6643 or Hu5F9) or anti-PD-L1 mAbs for 30-minutes. Then, PBMCs were added and incubated for 90 minutes at 37°C. After incubation, cells were resuspended in FACS buffer containing anti-human CD14-PE (BioLegend, Cat. 325606) antibody and incubated for 15 minutes at 4°C, washed, fixed, and analyzed by flow cytometry as above.

Thaker Y.R., Rivera I., Pedros C., Singh A.R., Rivero-Nava L., Zhou H., Swanson B.A., Kerwin L., Zhang Y., Gray J.D., Kaufmann G.F., Ji H., Allen R.D, & Bresson D. (2022). A Novel Affinity Engineered Anti-CD47 Antibody With Improved Therapeutic Index That Preserves Erythrocytes and Normal Immune Cells. Frontiers in Oncology, 12, 884196.

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