The monocyte isolation procedure was the same as in a previous method.39 (link) In brief, blood samples were diluted in saline at a 1:10 ratio, and then the mixture was further incubated with an OptiPrep gradient medium (07820; StemCell Technologies, Cambridge, MA, USA) containing Ficoll. After centrifuging at 700 × g for 20 min, the peripheral blood mononuclear cells (PBMCs) were collected. The CD142+monocytes were prepared from PBMCs with magnetic beads coated with anti-CD14 antibody (11149D; Thermo Fisher Scientific, Waltham, MA, USA). The purity of the monocytes was determined using flow cytometry with an anti-CD14 antibody (12014942; Invitrogen, Carlsbad, CA, USA).
Anti cd14 antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-CD14 antibody is a laboratory reagent used to detect and quantify the expression of the CD14 cell surface receptor. CD14 is a glycosylphosphatidylinositol-anchored protein expressed primarily on the surface of monocytes and macrophages, and plays a role in the innate immune response to bacterial lipopolysaccharide. The Anti-CD14 antibody can be used in various immunological techniques, such as flow cytometry, to study the expression and function of CD14 in biological samples.
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Lab products found in correlation
Fluorescein isothiocyanate (fitc), by Merck Group (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Sirnas, by Thermo Fisher Scientific (1 mentions)
Hiperfect transfection reagent, by Qiagen (1 mentions)
Goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Accurie c6 flow cytometer, by BD (1 mentions)
Rpmi medium, by Merck Group (1 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Zen software, by Zeiss (1 mentions)
5 protocols using anti cd14 antibody
1
Monocyte Isolation from Peripheral Blood
2
Isolation and Knockdown of SLAMF7 in CD14+ Myeloid Cells
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CD14+ myeloid cells (i.e. monocytes and macrophages) were isolated from human atherosclerotic plaques using magnetic beads bound to anti-CD14 antibody (Invitrogen, USA), as previously described [32 (link)]. Alternatively, peripheral blood monocytes (PBMCs) were separated by density gradient centrifugation of healthy donors. These cells were cultured overnight in fully supplemented DMEM in a 37°C 5% CO2 incubator. siRNAs targeting SLAMF7 (Invitrogen, USA) and HiPerFect transfection reagent (Qiagen, USA) were used to knockdown the protein expression of SLAMF7 in the cultured CD14+ cells. Conditioned media was collected from cultured cells, stimulated with 10 ng/mL of LPS for 24h [33 (link)]. Human aortic smooth muscle cell line was purchased from LifeLine Cell Technology (Shanghai, China).
3
Immunophenotyping of Bovine Leukocytes
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The RPMI medium and the Fluoresceinisothiocyanate were acquired from Sigma-Aldrich (FITC, Sigma-Aldrich, St. Louis, MO, USA). The vacutainer tubes containing EDTA were acquired from Becton Dickinson (BD Heidelberg, Germany. The antibodies used for cell labeling are described in Table 1 . The anti-CD14 antibody and the goat anti-mouse secondary antibodies were acquired from Thermofisher Scientific (Thermofisher Scientific, Hennigsdorf, Germany. The anti-MHCII and anti-CD163 antibodies were acquired from Kingfisher Biotech (Kingfisher Biotech, Saint Paul, Minnesota, USA). The antibodies to CD172a and CD11a were purchased from the monoclonal antibody center of the Washington State University (WSU, Pullman, Washington, USA). The anti-CD4 and anti-WC1 antibodies were acquired from XCeltis (XCeltis GmbH, Mannheim, Germany). Heat-killed staphylococcus aureus was purchased from Calbiochem (Pansorbin, Calbiochem, Merck, Nottingham, UK). Dihydrorhodamine-123 was acquired from Mobitec (Mobitec, Goettingen, Germany). The Accurie C6 flow cytometer was acquired from BD Biosciences (BD Biosciences, San Jose, CA, USA).
4
Immunohistochemical Analysis of CD14 Expression in Rat Liver Tissue
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Rat liver tissue samples were fixed in formaldehyde solution (10%, w/v), dehydrated with ethanol solutions of differing concentrations (50–100%, v/v), embedded in paraffin molds, cut into 4-μm thick sections, and stained for CD14 expression. Immunohistochemical staining was conducted following previously described standard procedures [61 (link)]. The standard procedure of antigen retrieval (citrate buffer) and endogenous peroxidase blockage (using 3% hydrogen peroxide) was performed prior to an overnight incubation with the primary rabbit polyclonal anti-CD14 antibody (1:60) (Thermo Fisher Scientific, Waltham, MA, USA) in a moist chamber. The visualization was effectuated using diaminobenzidine and counterstained with Mayer’s hematoxylin. Pathohistological analysis of the stained slides was performed on a light microscope (Olympus BX43, Olympus Corporation, Tokyo, Japan) [61 (link)]. Immunohistochemical staining was semi-quantitatively graded as: absent/no staining (0), trace (0.5), mild (1), moderate (2), and strong positive staining (3) [61 (link)].
5
Uptake of Fluorescent Nanoparticles in Monocytes
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Heparinized human whole blood samples were incubated with 6-FAM-labeled PFOB-NE with or without presence of Cytochalasin D (10 µM) at a dilution of 1:20 at 37 °C under constant shaking. After 128 min a probe of 500 µL of whole blood was taken, placed on ice and lysed three times for 15 min. The cells were fixed with PFA (1%) for 10min. After three washing steps, unspecific binding was blocked with BSA (3%, 1 h). Cells were immunostained using anti-CD14 antibody (15µg/mL, Thermo Fisher Scientific) for 12 h, followed by incubation with Alexa594-labeled secondary antibody (Thermo Fisher Scientific). The cells were washed and mounted with ProLong™ Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific). For visualization of ingested 6-FAM-labeled PFOB-NE, cells were analyzed by confocal microscopy (LSM700, Carl Zeiss Microscopy GmbH, Jena, Germany) using ZEN software (Version 2.3, Carl Zeiss Microscopy GmbH, Jena, Germany).
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