Hla dr pecy7

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The HLA-DR PECy7 is a fluorochrome-conjugated antibody used for the identification and enumeration of human leukocytes expressing the HLA-DR antigen. It is designed for use in flow cytometry applications.

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HLA-DR (57 citations)

7 protocols using hla dr pecy7

Multiparametric Immune Cell Profiling

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1 × 10⁶ cells were Fc blocked (Biolegend101320) and stained for 1 hour, on ice, in the dark, as previously described.^{20 (link)} The following antibodies were used, B220−FITC (BD553087), I Ab PerCP−Cy5.5 (Biolegend116416), Ly6C PE or PECy7 (Biolegend128008, 128071), HLA−DR PECy7 (eBioscience25−9956−41/Biolegend307606), CD11b BV510 (BD562950/Biolegend101263), Ly6G BV605 (BD563005/Biolegend127639), CD11c APC (BD550261), CD3 A700 (eBioscience56−0032−82/Biolegend100216), Live Blue Fluorescent reactive dye (LifeTech L34962), and CD45 BV650 (Biolegend103151).

Jankowska−Gan E., Agashe V.V., Lema D.A., Zhou Y., Gonzalez Bosc L., Sullivan J.A., Greenspan D.S, & Burlingham W.J. (2020). Donor HLA−DR Drives the Development of De Novo Autoimmunity Following Lung and Heart Transplantation. Transplantation Direct, 6(10), e607.

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Multiparametric Flow Cytometry Analysis

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PBMCs (1 × 10⁶) were Fc blocked (130-059-901; Miltenyi Biotec), stained, and acquired on a BD LSR II or a BD LSRFortessa (5 (link)). For intracellular cytokine staining, 1 × 10⁶ PBMCs were cultured in the presence of the appropriate antiegn (ColI or ColV) in DMEM containing 5% FBS at 37°C. After overnight incubation, the cells were stimulated with PMA (10 ng/ml) and ionomycin (1 μg/ml) for 5 h, with the addition of brefeldin A (420601; BioLegend) for the last 3 h. The following Abs were used: CD3-FITC or CD3–Alexa Fluor 700 (317306/100216; BioLegend or 557943; BD Biosciences), CD11b-BV421 (562632; BD Biosciences), CD56-allophycocyanin (555518; BD Biosciences), CD14–Alexa Fluor 700 (325614; BioLegend), CD16-allophycocyanin-H7 (560195; BD Biosciences), LAIR1-PE (550811; BD Biosciences), HLA-DR–PE-Cy7 (25-9956-41; eBioscience), CD123-BV421 (306017; BioLegend), lineage-BV510 (348807; BioLegend), CD11c-allophycocyanin (559877; BD Biosciences), PE-IgG₁ κ isotype control (55749; BD Biosciences), CD196/CCR6-FITC (11-1969-42; eBioscience), CD194/CCR4-PECy7 (561034; BD Biosciences), CD4-BUV395 (563552/563790; BD Biosciences), CD183/CXCR3-allophycocyanin (550967; BD Biosciences), IFN-γ-BV421 (563376; BD Biosciences), IL-17-PE (559502; BD Biosciences), γδ TCR-FITC (118105; BioLegend). Data were analyzed using FlowJo (Treestar).

Agashe V.V., Jankowska-Gan E., Keller M., Sullivan J.A., Haynes L.D., Kernien J.F., Torrealba J.R., Roenneburg D., Dart M., Colonna M., Wilkes D.S, & Burlingham W.J. (2018). Leukocyte-Associated Ig-like Receptor 1 Inhibits Th1 Responses but Is Required for Natural and Induced Monocyte-Dependent Th17 Responses. Journal of immunology (Baltimore, Md. : 1950), 201(2), 772-781.

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PBMC Isolation and B Cell Sorting

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Peripheral blood mononuclear cells (PBMCs) were obtained from blood. After Ficoll-Isopaque density centrifugation (Rafer, Zaragoza, Spain), collected cells were washed twice with ice-cold PBS, followed by centrifugation at 2000 rpm for 5 min. Next, cells were labeled with antibodies to CD19 – FITC (Miltenyi Biotec, clone LT19), CD27 – APC (Miltenyi Biotec, clone M-T271), IgD – PE (SouthernBiotech, Cat. No. 2032-09) and IgM – PerCP/Cy5.5 (BioLegend, clone MHM-88) for 20 min on ice in a staining buffer (PBS with 4% FBS and 2 mM EDTA). Naïve B cells (CD19⁺ CD27^– IgD⁺) and unswitched memory B cells (CD19⁺ CD27⁺ IgD⁺) were obtained by FACS sorting on a MoFlo Astrios (Beckman Coulter). Purified samples were pelleted and stored at −80°C.
For isolation of naïve autoreactive B cells. Total B cells were isolated from PBMCs using positive selection with MACS CD19 microbeads (Miltenyi Biotec). Next, cells were stained with CD27-APC (Miltenyi Biotec, clone M-T271), IgD – PE (SouthernBiotech, Cat. No. 2032-09), HLA-DR – PE-Cy7 (eBioscience, clone LN3), 9g4 primary ab (igm Bioscience) and donkey anti-rat IgG (H + L) – Alexa Fluor 488 (invitrogen). 9g4+ naïve B cells (CD27^– IgD⁺ 9g4⁺) and 9g4- naïve B cells (CD27^– IgD⁺ 9g4^–) were obtained by FACS sorting on a BD FACSAria II (BD Biosciences). Purified samples were pelleted and stored at −80°C.

Català-Moll F., Ferreté-Bonastre A.G., Li T., Weichenhan D., Lutsik P., Ciudad L., Álvarez-Prado Á.F., Rodríguez-Ubreva J., Klemann C., Speckmann C., Vilas-Zornoza A., Abolhassani H., Martínez-Gallo M., Dieli-Crimi R., Rivière J.G., Martín-Nalda A., Colobran R., Soler-Palacín P., Kracker S., Hammarström L., Prosper F., Durandy A., Grimbacher B., Plass C, & Ballestar E. (2021). Activation-induced deaminase is critical for the establishment of DNA methylation patterns prior to the germinal center reaction. Nucleic Acids Research, 49(9), 5057-5073.

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Comprehensive Cervical Cell Phenotyping

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The phenotype of cells collected on the cervical brushes was determined using flow cytometry. Squamous epithelial cells and dead leukocytes absorbed 4′,6-diamino-2-phenylindole (DAPI). Cells that excluded DAPI were examined using directly labelled monoclonal antibodies against the following human proteins: CD45-FITC and CD16-PerCPCy5.5 from BD Biosciences (Franklin Lakes, NJ, USA), CD163-PE, CD11b-PE, CD20-PE, CD14-PerCPCy5.5, HLA-DR-PECy7, CD19-PECy7, CD27-PECy7, CCR7-PECy7, CD11b-APC (activation epitope CBRM1/5) and CD16-APC from eBioscience (San Diego, CA, USA); CD235a-PE, CD68-PE, CD66b-PE, CD103-PE, CD11c-PE, CX3CR1-PE, CCR2-PerCPCy5.5, CD3-PECy7, CCR1-APC, CD4-APC, CD33-APC and CD64-APC from Biolegend (San Diego, CA, USA). Flow cytometric data were collected on an LSRII (BD Biosciences). A minimum of 300,000 events were collected for each group of antibodies so that even a leukocyte population consisting of 0.01% of the total host-derived cells could be reliably detected. Data were analysed using FlowJo 9.6.4 (TreeStar, Ashland, OR, USA).

Hunter P.J., Sheikh S., David A.L., Peebles D.M, & Klein N. (2016). Cervical leukocytes and spontaneous preterm birth. Journal of Reproductive Immunology, 113, 42-49.

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Ex vivo Flow Cytometry Analysis of PBMCs

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For ex vivo flow cytometry analysis, PBMCs were thawed and stained with viability dye (eFluor 780; eBioscience). Purified anti-FCRL3 (24 (link)) was used where mentioned, followed by staining with F(ab′)₂ anti-mouse IgG (eFluor 660 or PE; eBioscience) and extensive washing with PBS. Cells were then stained with fluorochrome-conjugated Abs against CD4 (FITC or V500), CD25 (PE-CF594), CD45RA (Alexa Fluor 700) (BD Biosciences), CD127 (PE), TIGIT (PerCP–eFluor 710), FOXP3 (PE-Cy7), CD62L (FITC), HLA-DR (PE-Cy7), CTLA-4 (allophycocyanin) (eBioscience), and Helios (Pacific Blue; BioLegend).
For cytokine detection, PBMCs were incubated with PMA (25 ng/ml), ionomycin (1 μg/ml) (both from Sigma-Aldrich), and GolgiStop (eBioscience) for 4 h, followed by intracellular cytokine staining with Abs against IFN-γ (FITC), IL-2 (PerCP-Cy5.5), and IL-17 (allophycocyanin) (eBioscience).
Flow cytometry analysis was performed on an LSRFortessa analyzer, and sorting throughout this study was performed on a FACSAria IIu cell sorter (both from BD Biosciences).

Dhuban K.B., d’Hennezel E., Nashi E., Bar-Or A., Rieder S., Shevach E.M., Nagata S, & Piccirillo C.A. (2015). Coexpression of TIGIT and FCRL3 Identifies Helios+ Human Memory Regulatory T Cells. Journal of immunology (Baltimore, Md. : 1950), 194(8), 3687-3696.

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Immune Cell Phenotyping and Functional Assays

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Mo/Ma were harvested and stained with anti-human CD14 AlexaFluor-647 or FITC, CD206 APC, Gal-9 PE, CD16 PE, CD40 PE-Cy5 (Biolegend), HLA-DR PE-Cy7, B7.H4 Biotin (eBioscience, San Diego, CA, USA) or isotype controls in PBS with 2% FSB on ice for 20 min. T cells were harvested and stained with mAb to human CD3 PerCP, CD27 APC-Alexa750, CD28 APC and Tim-3 PerCP-eFluor710 (eBioscience).
To detect CD40L expression, PE-labeled anti-human CD40L/CD154 antibodies were cultured with T cells in the presence of anti-CD3 mAb and Monensin solution (eBioscience) during 40 h. Intracellular staining for phosphotyrosine was performed an AlexaFluor-647-conjugated anti-human phosphotyrosine (PY20, Biolegend) antibody.
The production of ROS and NO was evaluated using the molecular probes: H₂DCF-DA (10 μM, Invitrogen Inc.) and DAF-FM DA (10 μM, Molecular Probes, Inc.), respectively.
The assessment of phagocytosis was performed using 1 μm-Fluoresbrite Yellow Green (YG) Carboxilate Microspheres (Polysciences, Inc., Warrington, PA, USA; 1 : 25 cell/microspheres ratio), which were added to CCs for 30 min. Afterwards, the uptake of YG-microspheres was evaluated in CD14+ cells.
All samples were acquired on a FACS Canto II (BD Biosciences, San Jose, CA, USA) and then analyzed with Flow Jo software (LLC, Ashland, OR, USA).

Ramello M.C., Tosello Boari J., Canale F.P., Mena H.A., Negrotto S., Gastman B., Gruppi A., Acosta Rodríguez E.V, & Montes C.L. (2014). Tumor-induced senescent T cells promote the secretion of pro-inflammatory cytokines and angiogenic factors by human monocytes/macrophages through a mechanism that involves Tim-3 and CD40L. Cell Death & Disease, 5(11), e1507-.

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Immunophenotyping of Differentiated Cells

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For the study of surface cell markers, cells were harvested after differentiation culture and washed once with PBS. Cell staining was performed in a staining buffer (PBS with 4% fetal bovine serum and 0.4% EDTA) after blocking for non-specific binding with Fc block (BD Pharmingen) for 5 minutes on ice. Cells were stained for 20 minutes on ice. Antibodies used included: CD14-FITC, CD80-PE, CD86-APC (Miltenyi biotec), CD11b-APC, CD1a-PE (Biolegend), HLA-DR-PeCy7 (eBioscience). Cells were also stained with the viability dye LIVE/DEAD TM Fixable Violet (Invitrogen) according to manuacturer's conditions. After staining, cells were fixed with PBS + 4% paraformaldehyde and analyzed in a BD FACSCanto-II flow cytometer in the following 48 h.

Català-Moll F., Ferreté-Bonastre A.G., Godoy-Tena G., Morante-Palacios O., Ciudad L., Barberà L., Fondelli F., Martínez-Cáceres E.M., Rodríguez-Ubreva J., Li T, & Ballestar E. (2022). Vitamin D receptor, STAT3, and TET2 cooperate to establish tolerogenesis. Cell reports, 38(3).

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