Cd14 m5e2

Manufactured by BioLegend

Sourced in United States

CD14 (M5E2) is a mouse monoclonal antibody that binds to the CD14 antigen, a glycosylphosphatidylinositol (GPI)-anchored protein expressed on the surface of monocytes and macrophages. CD14 functions as a pattern recognition receptor that binds to lipopolysaccharide (LPS) and other pathogen-associated molecular patterns (PAMPs), playing a key role in the innate immune response to bacterial infection.

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Lab products found in correlation

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Close spelling variants of this product

Anti-CD14-Alexa Fluor 647 (clone M5E2, APC-cy7 anti-human CD14 (clone M5E2; CD14 BV510 (M5E2, CD14-FITC (clone M5E2), CD14-BV785 clone M5E2 CD14 PE-Cy7 (M5E2, CD14 APC-Cy7 (M5E2; PE-conjugated anti-CD14 mouse IgG2a (M5E2; Anti-human CD14-FITC (clone M5E2, CD14 (clone M5E2,

17 protocols using cd14 m5e2

Phenotypic Characterization of Immune Cells

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Monocyte purity, proper Mo-DC differentiation and the basal state of activation of both cells were assessed by flow cytometry using the following anti-human monoclonal antibodies: CD14 (M5E2, Biolegend, San Diego, CA, USA), CD1b (SN13, Biolegend), CD1d (51.1, Biolegend), CD11c (3.9, eBioscience), CD80 (2D10, Biolegend), HLA-DR (LN3, eBioscience).
iNKT and T cell determinations were performed in total PBMCs or in CD14⁻ fractions, using CD1d-PBS57 tetramers (NIH Tetramer Core Facility, Emory University, Atlanta, GA, USA) and the following anti-human monoclonal antibodies: CD3 (OKT3, eBioscience), CD4 (OKT4, Biolegend), and CD8 (RPA-T8, eBioscience). The purity of T cell clones VM-D5 and JS63 was assessed by using CD1d-PBS57 tetramers (NIH Tetramer Core Facility, Emory University, Atlanta, GA, USA) together with anti-human CD3 (OKT3, eBioscience) monoclonal antibody. The purity of T cell clones s33d, GG33A, and DS1C9b was assessed by using anti-human TCR Vβ13.1 (IMMV222), Vβ18 (BA62.6), and Vβ7.1 (ZOE), monoclonal antibodies from Immunotec (Immunotec Research Inc, Canada). Cells were acquired in a FACS Canto II (BD Biosciences, San Diego, CA, USA) using the BD FACSDiva™ software (BD Biosciences). Data analysis was performed with FlowJo® v10 (FlowJo LLC, Ashland, OR, USA).

Pereira C.S., Pérez-Cabezas B., Ribeiro H., Maia M.L., Cardoso M.T., Dias A.F., Azevedo O., Ferreira M.F., Garcia P., Rodrigues E., Castro-Chaves P., Martins E., Aguiar P., Pineda M., Amraoui Y., Fecarotta S., Leão-Teles E., Deng S., Savage P.B, & Macedo M.F. (2019). Lipid Antigen Presentation by CD1b and CD1d in Lysosomal Storage Disease Patients. Frontiers in Immunology, 10, 1264.

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Multiparameter Immune Cell Profiling

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CD45 (HI30); MerTK (2B10C42); CD14 (M5E2); CD64 (10.1); CCR2 (K036C2); HLA-DR (L243).
All antibodies listed above were purchased from Biolegend with the exception of CCR2 (475301) (R&D Systems), Lyve1 (ALY7) (eBiosciences) and B220 (RA3–6B2); BV421-SA (BD Biosciences). Antibody clone is provided in parentheses. For more details see the Life Sciences Reporting Summary

Dick S.A., Macklin J.A., Nejat S., Clemente-Casares X., Momen A., Kantores C., Hosseinzadeh S., Barbu I., Chen J., Althagafi M.G., Besla R., Wong A., Aronoff L., Zaman R., Lavine K.J., Razani B., Ginhoux F., Husain M., Cybulsky M.I., Robbins C.S, & Epelman S. (2018). Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction. Nature immunology, 20(1), 29-39.

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Comprehensive Immune Cell Profiling

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Cells harvested after overnight incubation were stained following standard protocols. Experiments used antibodies specific for human FcR block (BioLegend #422302), CD11c (Bu15, BioLegend #337214), CD80 (2D10, BioLegend #305220), CD86 (Bu63, BioLegend #374210) HLA-DR (L243, BioLegend #307629), HLA-A,B,C (W6/32, BioLegend #311410), CD14 (M5E2, BioLegend #301828), CD63 (H5C6, BioLegend #353007), CD61 (VI-PL2, BioLegend #336416), CD41/CD61 (PAC-1, BioLegend 362804), CD62p (AK4, BioLegend #304906), mTCRb (H57-597, BioLegend #109206), CD4 (OKT4, BioLegend #317428), CD8 (SK1, BioLegend #344704), and human CD3-APC (Clone OKT3, BioLegend). Color-matched isotype control antibodies were obtained from the same vendors. Flow cytometry was performed with a Stratedigm flow cytometer with electronic gates set on live cells by a combination of forward and side light scatter and 7-AAD (BioLegend), EMA (Invitrogen), Zombie Aqua (BioLegend), or Zombie NIR (BioLegend) exclusion. A minimum of 3 × 10⁴ events were collected per sample, and data were analyzed with FlowJo software (FlowJo LLC).

Han P., Hanlon D., Arshad N., Lee J.S., Tatsuno K., Robinson E., Filler R., Sobolev O., Cote C., Rivera-Molina F., Toomre D., Fahmy T, & Edelson R. (2020). Platelet P-selectin initiates cross-presentation and dendritic cell differentiation in blood monocytes. Science Advances, 6(11), eaaz1580.

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Multiparametric Immunofluorescence Profiling

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Frozen skin sections were fixed with acetone and blocked with 10% normal goat serum (Vector Laboratories) for 30 minutes. Primary antibodies were incubated overnight at 4°C and amplified with the appropriate Alexa Fluor® 488 (A-488) or 568 (A-568) conjugated secondary antibody for 30 minutes at room temperature. Antibodies used are: IL-32αβγδ (KU32–52, BioLegend), CD3 (SK7, BD Biosciences), CD4 (SK3, BD Biosciences), CD8 (SK1, BD Biosciences), hNKp46 (195314, R&D Systems), CD20 (L27, BD Biosciences), CD14 (M5E2, BioLegend), CD11c (B-ly6, BD Pharmingen), CD303 (AC144, Miltenyi Biotec), and CD163 (5C6-FAT, Acris Antibodies).

Ohmatsu H., Humme D., Gulati N., Gonzalez J., Möbs M., Suárez-Fariñas M., Cardinale I., Mitsui H., Guttman-Yassky E., Sterry W, & Krueger J.G. (2014). Interleukin-32 is progressively expressed in Mycosis Fungoides independent of helper T-cell 2 and helper T-cell 9 polarization. Cancer immunology research, 2(9), 890-900.

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Phenotypic Profiling of Monocytes and DCs

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1x10⁶ cryopreserved PBMC were thawed then washed twice in FACS buffer and surface stained using the following antibody cocktail: CD14 (M5E2, Biolegend), CD16 (3G8, Biolegend), CD11b (ICRF44, Biolegend), HLA-DR (L243, Biolegend), CD40 (5C3, BD Pharmingen), CD62L (DREG-56, BD Pharmingen), CD64 (10.1, Biolegend), CD163 (GHI/61, Biolegend), CXCR6 (K041E5, Biolegend), IFNAR (85228, R&D Systems), CD11c (3.9, ThermoFisher Scientific) and CD123 (6H6, Biolegend) for the detection of various monocytic and dendritic cell subsets. Samples were then acquired on the Attune NxT acoustic focusing cytometer (Life Technologies). Data were analyzed using FlowJo v10 (TreeStar, Ashland, OR USA).

Zulu M.Z., Sureshchandra S., Pinski A.N., Doratt B., Shen W, & Messaoudi I. (2021). Obesity Correlates With Pronounced Aberrant Innate Immune Responses in Hospitalized Aged COVID-19 Patients. Frontiers in Immunology, 12, 760288.

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Flow Cytometric Profiling of PBMCs

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Frozen PBMCs were thawed, washed in FACS buffer (2% FBS, 1mM EDTA in PBS), and counted on TC20 (Biorad) before surface staining using two independent flow panels. For the innate panel, the following antibodies were used: CD3 (SP34, BD Pharmingen) and CD20 (2H7, BioLegend) for the exclusion of T & B lymphocytes, respectively. We further stained for CD56 (HCD56, Biolegend), CD57 (HNK-1, BioLegend), KLRG1 (SA231A2, BioLegend) CD16 (3G8, BioLegend), CD14 (M5E2, BioLegend), HLA-DR (L243, BioLegend), CD11c (3.9, ThermoFisher Scientific), CD123 (6H6, BioLegend) and CD86 (IT2.2, BioLegend). For the adaptive panel, the following antibodies were used: CD4 (OKT4, BioLegend), CD8b (2ST8.5H7, Beckman Coulter), CD45RA (HI100, TONBO Biosciences), CCR7 (G043H7, BD Biosciences), CD19 (HIB19, BioLegend), IgD (IA6–2, BioLegend), CD27 (M-T271, BioLegend), KLRG1 (SA231A2, BioLegend) and PD-1 (Eh12.2h7, BioLegend). Cells were stained with Ghost Dye viability dye (TONBO biosciences) for 30 minutes at 4C per manufacturer’s instructions, washed, surface stained with either innate or adaptive panels for 30 minutes at 4C. Samples were then washed and analyzed on Attune NxT Flow Cytometer (ThermoFisher Scientific, Waltham MA).

Sureshchandra S., Zulu M.Z., Doratt B., Jankeel A., Tifrea D., Edwards R., Rincon M., Marshall N.E, & Messaoudi I. (2021). Deep immune profiling of the maternal-fetal interface with mild SARS-CoV-2 infection. bioRxiv.

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Dendritic Cell Differentiation Protocol

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RPMI 1640 and low-endotoxin FBS were obtained from Lonza (Walkersville, MD, USA). Recombinant human GM-CSF and human IL-4 were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany); Flow cytometric analysis was performed using the following mouse anti-human antibodies: CD83 (HB15e) and CD1a (HI149) (Becton Dickinson, San Jose, CA, USA); CD80 (2D10), CD86 (T2.2), HLA-DR (L243) and CD14 (M5E2) (Biolegend, San Diego, CA, USA). PLGA (poly[DL-lactide-co-glycolide] 50:50 lactide-glycolide ratio, CAS 26780-50-7), PVA (poly[vinyl alcohol], CAS 9002-89-5), Acetone (≥99% purity, 1.00013), Dimethyl sulfoxide (DMSO, ≥99% purity D-5879), Oxyresveratrol (≥97% purity, 91211) were purchased from Sigma-Aldrich, (St. Louis, MO, USA).

Gaglio S.C., Donini M., Denbaes P.E., Dusi S, & Perduca M. (2021). Oxyresveratrol Inhibits R848-Induced Pro-Inflammatory Mediators Release by Human Dendritic Cells Even When Embedded in PLGA Nanoparticles. Molecules, 26(8), 2106.

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Degranulation Assay for NK and CD8+ T Cells

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Degranulation of human NK cells and CD8⁺ T cells were performed as following; 3×10⁵ PBMC were incubated for 3 hours in vitro alone, in presence of 1:1 K562 cells, or with phorbol 12-myristate 13-acetate (50 ng/mL; Sigma-Aldrich), and Ionomycin (250 ng/mL; Sigma-Aldrich). Cells were then stained for surface staining including CD3 (SK7), CD8 (RPA-T8), CD56 (MEM188), CD16 (CB16), CD107a (LAMP-1) and CD107b (H4B4) (all from Invitrogen, Thermofisher) and CD14 (M5E2, from Biolegend) and analyzed by flow cytometer CANTO II (BD Bioscience).

Humblet-Baron S., Franckaert D., Dooley J., Ailal F., Bousfiha A., Deswarte C., Oleaga-Quintas C., Casanova J.L., Bustamante J, & Liston A. (2018). IFN-γ and CD25 drive distinct pathological features during hemophagocytic lymphohistiocytosis. The Journal of allergy and clinical immunology, 143(6), 2215-2226.e7.

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Ex vivo Phosphorylation Assays of PBMCs

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For ex vivo phosphorylation assays, thawed PBMC were plated at 250,000 cells per well in 96 well polypropylene plates and rested for 2 h at 37 °C, 5% CO₂. PBMC were fixed with pre-warmed (37 °C) Cytofix (BD Biosciences) for 20 min at 37 °C, 5% CO₂ and permeabilized with Perm III buffer (BD Biosciences) overnight at −20 °C. Cultures were subsequently stained with CD3 (UCHT1, BD Biosciences), CD20 (H1, BD Biosciences), CD14 (M5E2, Biolegend), CD16 (B73.1, BD Biosciences), phospho-IRF3 (Ser 396, Bioss), phospho-NFkB p65 (Ser 529, BD Biosciences) in PBS for 1 hour at room temperature, washed with PBS and resuspended in 250 μl PBS.
For phosphorylation assays after LPS stimulation, PBMC were plated as above and stimulated with 100 ng/ml LPS for a total of 1 hour. Samples were fixed at 0, 5, 15, 30, 45, and 60 min after LPS addition for 20 min at 37 °C, 5% CO₂, and stained as above.

Maher A.K., Burnham K.L., Jones E.M., Tan M.M., Saputil R.C., Baillon L., Selck C., Giang N., Argüello R., Pillay C., Thorley E., Short C.E., Quinlan R., Barclay W.S., Cooper N., Taylor G.P., Davenport E.E, & Dominguez-Villar M. (2022). Transcriptional reprogramming from innate immune functions to a pro-thrombotic signature by monocytes in COVID-19. Nature Communications, 13, 7947.

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Bacterial Agonist-Induced PBMC Cytokine Response

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1x10⁶ cryopreserved PBMC were thawed, washed, and then stimulated with a bacterial agonist cocktail or left unstimulated. The bacterial agonist cocktail consisted of a combination of 2ug/mL Pam3CSK4 (TLR1/2 agonist, InvivoGen), 1ug/mL FSL-1 (TLR2/6 agonist, Sigma Aldrich), and 1ug/mL LPS (TLR4 agonist from E. coli 0111: B4, InvivoGen). Samples were cultured for 1 hour before adding protein transport inhibitor (Brefeldin A) and incubated for an additional 7 hours at 37C. Cells were then washed twice in FACS buffer and surface stained using the following antibody cocktail – CD14 (M5E2, Biolegend) and HLA-DR (L243, Biolegend) for 30 minutes at 4C. Stained cells were then fixed and permeabilized using Fixation buffer (Biolegend) and incubated overnight with a cocktail of intracellular antibodies – IL-6 (MQ2-6A3, Biolegend), and TNFɑ (Mab11, eBioScience). Samples were then acquired on the Attune NxT acoustic focusing cytometer (Life Technologies). Data were analyzed using FlowJo v10 (TreeStar, Ashland, OR USA).

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