Anti hla dr pe

Manufactured by Beckman Coulter

Sourced in United States

The Anti-HLA-DR-PE is a fluorescent-labeled monoclonal antibody that binds to the HLA-DR antigen expressed on the surface of various cell types, including antigen-presenting cells. It is used in flow cytometry applications to identify and characterize HLA-DR-positive cells.

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5 protocols using anti hla dr pe

Flow Cytometry Analysis of Cell Markers

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Flow cytometry was performed as previously described [26 (link),27 (link)]. Analysis of the expression levels of cell-surface markers was performed by flow cytometry. DCs were blocked for 5 min with PBS containing 10% heat inactivated human AB serum (Sigma-Aldrich, St. Louis, MO, USA), and then stained by adding the antibodies to the same buffer. All other cell types were stained on PBS containing 3% FBS. Cells were labeled with anti-CD1a-PE, anti-CD3-PC7, anti-CD4-FITC, anti-CD14-PC7, anti-CD19-ECD, anti-CD25-PC7, anti-CD69-PC5, anti-CD80-FITC, anti-CD86-PC5.5, anti-CD86-PE, anti-HLA-DR-ECD, anti-HLA-DR-PB, anti-HLA-DR-PE (all from Beckman Coulter Inc., Brea, CA, USA), anti-CD8-Pacific Blue, anti-CD71-APC-Cy7, anti-CD197-APC-Cy7 (CCR7) (all from BioLegend, San Diego, CA, USA), anti-CD83-APC (BD Biosciences, Franklin Lake, NJ, USA), and anti-CD3-PO (Abcore, Ramona, CA, USA).
Levels of surface expression on cells were estimated by flow cytometry (Gallios; Beckman Coulter, Brea, CA, USA) and analyzed using Kaluza software (Beckman Coulter, Brea, CA, USA). Entry of FITC-labeled p24 peptide into the DCs was also estimated by flow cytometry. DCs were collected 2 h, 24 h, and 48 h after complex addition, washed with PBS, and acid washed, in order to eliminate any peptide attached to the cell surface. Fluorescence was measured by flow cytometry.

Martín-Moreno A., Jiménez Blanco J.L., Mosher J., Swanson D.R., García Fernández J.M., Sharma A., Ceña V, & Muñoz-Fernández M.A. (2020). Nanoparticle-Delivered HIV Peptides to Dendritic Cells a Promising Approach to Generate a Therapeutic Vaccine. Pharmaceutics, 12(7), 656.

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Isolation and Characterization of NK Cells and PMN-MDSCs

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NK cells and PMN-MDSCs were isolated from PBMCs of mobilized and non-mobilized donors using NK isolation kit and CD66b⁺ microbeads (purity >98%, data not shown) following manufacturer’s instruction (Miltenyi Biotec, Bergisch Gladbach, Germany). Before starting any experiment, we determined the purity of isolated cells by flow cytometry using anti-CD3-APC, anti-CD19-ECD, and CD56-PC7 (Beckman Coulter, Brea, CA) for NK cells. PMN-MDSCs were labeled with anti-CD3-AF700, anti-CD19-AF700, anti-CD11b-FITC, anti-CD33-PC7, anti-HLA-DR-PE, anti-CD14-ECD, anti-CD45-KrOr, and anti-CD66b-APC desiccated in the Duraclone custom design platform (Beckman Coulter, Brea, CA) adding anti-CD56-BV650 (BioLegend, San Diego, CA) and following manufacturer’s instruction. After the staining procedures, cells were acquired at Cytoflex LX and analyzed with Cytexpert software (v2.4, Beckman Coulter, Brea, CA). Freshly isolated NK cells were immediately used for functional studies and gene expression evaluation.

Pelosi A., Besi F., Tumino N., Merli P., Quatrini L., Li Pira G., Algeri M., Moretta L, & Vacca P. (2021). NK Cells and PMN-MDSCs in the Graft From G-CSF Mobilized Haploidentical Donors Display Distinct Gene Expression Profiles From Those of the Non-Mobilized Counterpart. Frontiers in Immunology, 12, 657329.

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T Lymphocyte Differentiation Profiling

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To define T lymphocytes differentiation status, both CTL and Th lymphocyte subsets were distinguished from CD3+CD8+ and CD3+CD4+ gate, respectively as follows: activated T lymphocytes (HLA-DR+), naïve (N; CD45RA+CD27+), central memory (CM; CD45RA−CD27+), effector memory (EM; CD45RA−CD27−) and effectors (E; CD45RA+CD27−) were evaluated using anti-CD45RA-FITC (L48, BD Bioscience, San Jose, CA, USA), anti-CD3-APC (SK7, BD Bioscience, San Jose, CA, USA), anti-CD8-APCH7 (SK1, BD Bioscience, San Jose, CA, USA), anti-CD4-V450 (RPA-T4, BD Bioscience, San Jose, CA, USA), anti-CD27-PerCP-Cy ™ 5.5 (L128, BD Bioscience, San Jose, CA, USA), and anti-HLA-DR-PE (L243, Beckman Coulter, Chaska, MN, USA). Gating strategies are shown in Supplementary Material. For each status (N, CM, EM, E, activated) the results were expressed as counts in percentage (%) in relation to the Th and the CTL.

Rios D.A., Casciato P.C., Caldirola M.S., Gaillard M.I., Giadans C., Ameigeiras B., De Matteo E.N., Preciado M.V, & Valva P. (2021). Chronic Hepatitis C Pathogenesis: Immune Response in the Liver Microenvironment and Peripheral Compartment. Frontiers in Cellular and Infection Microbiology, 11, 712105.

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Quantification of Serum Biomarkers and Immune Cell Phenotypes

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Blood was centrifuged and serum was stored in -80 °C until assayed. Levels of biomarkers were quantified by using commercially available kits of enzyme immunosorbent assays from Bio-Techne (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The lower limits of detection were: 16 pg/ml for tumor necrosis factor-alpha (TNFα); 40 pg/ml for IL-6; 31 pg/ml for IL-10; 62 pg/ml for IL-38; 156 pg/ml for IFNγ; and 313 p/ml for platelet-derived growth factor (PDGF)-A.
White blood cells were incubated for 15 minutes in the dark with the monoclonal antibodies anti-CD14 FITC, anti-HLA-DR-PE, anti-CD45 PC5 (Beckman Coulter, Marseille, France). White blood cells were also incubated for 15 minutes in the dark with anti-CD3 FITC, anti-CD4 FITC and anti-CD19 FITC (fluorescein isothiocyanate, emission 525nm, Beckman Coulter); with anti-CD4 PE, anti-CD8 PE, and anti-CD(16+56) PE (phycoerythrin, emission 575nm, Beckman Coulter); and with anti-CD45 PC5 (emission 667nm, Beckman Coulter). Fluorospheres (Beckman Coulter) were used for the determination of absolute counts. Cells were analyzed after running through the CYTOMICS FC500 flow cytometer (Beckman Coulter Co, Miami, Florida). Isotypic IgG controls stained also with anti-CD45 were used for each patient. Results of HLA-DR on CD14/CD45-cells were expressed as mean fluorescence intensity (MFI).

Bacharaki D., Karagiannis M., Giannakopoulos P., Papachristou E., Divanis D., Sardeli A., Petrou D., Nikolopoulos P., Bratsiakou A., Zoi V., Piliouras N., Damoraki G., Liakopoulos V., Goumenos D, & Giamarellos-Bourboulis E.J. (2023). Immune responses of patients on maintenance hemodialysis after infection by SARS-CoV-2: a prospective observational cohort study. BMC Infectious Diseases, 23, 581.

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Immunophenotyping of Cell Suspensions

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Cell suspension (1–2 × 10⁶) was incubated with antibodies for 20 min at 4 °C in 100 µL of phosphate buffered saline (PBS). The following anti-human monoclonal antibodies, all fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)- or allophycocyanin (APC)-conjugated, were used at 1:10 dilution: anti-CD90 FITC (ref: IM1839U), anti-CD73 PE (ref: B68176), anti-CD105 PC7 (ref: B43293), anti-CD45 FITC (ref: IM0647), anti-CD34 FITC (ref: IM1870), anti-CD14 FITC (ref: B36297), anti-HLA-DR PE (ref: IM1639), anti-CD19 APC (ref: IM2470), anti-CD31 FITC (ref: IM1431U) (Beckman Coulter, Brea, CA, USA), anti-CD146 APC (clone: REA773), anti-SUSD2 APC (clone: W5C5), and anti-EPCAM FITC (clone: HEA-125) (Miltenyi Biotech, Bergisch Gladbach, Germany). As negative control, cells were incubated without antibodies. Labelled cells were washed with PBS and analyzed using Navios cytometer (Beckman Coulter, CA, USA).

Canosa S., Mareschi K., Marini E., Carosso A.R., Castiglia S., Rustichelli D., Ferrero I., Gennarelli G., Bussolati B., Nocifora A., Asnaghi V., Bergallo M., Isidoro C., Benedetto C., Revelli A, & Fagioli F. (2022). A Novel Xeno-Free Method to Isolate Human Endometrial Mesenchymal Stromal Cells (E-MSCs) in Good Manufacturing Practice (GMP) Conditions. International Journal of Molecular Sciences, 23(4), 1931.

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