Hla dr fitc

Manufactured by Thermo Fisher Scientific

Sourced in United States

The HLA-DR-FITC is a fluorescently labeled antibody that binds to the HLA-DR antigen, which is expressed on the surface of various cell types, including antigen-presenting cells. This product is designed for use in flow cytometry applications to identify and quantify HLA-DR-positive cells in biological samples.

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Anti-HLA-DR-FITC HLA-DR

17 protocols using hla dr fitc

Phenotypic Characterization of MSCs and Monocytes

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For phenotypic characterization of MSCs and monocytes, samples were taken from both populations at times 0, 7, 14, and 28 days. MSCs were lifted with trypsin and monocytes with a scraper, and, after centrifugation at 300× g for 5 min at 4 °C, each cell type was resuspended in PBS. Monocytes were permeabilized with perm/wash buffer (BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 30 min at 4 °C with the following fluorochrome-conjugated antibodies: CD68-FITC, CD163-PE, and CD206-APC (BD Biosciences, Franklin Lakes, NJ, USA).
MSCs were incubated identically with the following fluorochrome-conjugated antibodies: CD73-APC, CD45-FITC, CD31-PE, CD90-APC, and HLA-DR-FITC (Invitrogen, Waltham, MA, USA).
Fluorescence-minus-one technique was used to adjust voltages and compensate for fluorescence, and propidium iodide (Sigma, Burlington, MA, USA) was used to determine viability following the manufacturer’s instructions.
A Guava EasyCyte flow cytometer (Merck, Darmstadt, Germany) was used to acquire the samples, and InCyte software (Merck, Darmstadt, Germany) was used to analyze the results.

Lapuente J.P., Blázquez-Martínez A., Marco-Brualla J., Gómez G., Desportes P., Sanz J., Fernández P., García-Gil M., Bermejo F., San Martín J.V., Algaba A., De Gregorio J.C., Lapuente D., De Gregorio A., Lapuente B., Andrés M.D, & Anel A. (2022). Cytokine Profile and Anti-Inflammatory Activity of a Standardized Conditioned Medium Obtained by Coculture of Monocytes and Mesenchymal Stromal Cells (PRS CK STORM). Biomolecules, 12(4), 534.

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Characterizing Surface Markers of DMCs

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The specific surface DMC molecules were characterized by flow cytometry analysis. Cells were dissociated by incubation with trypsin and were conjugated with the followed specific cell surface markers: CD44+, CD73+, CD29+, CD105+, CD90+, HLA-G+, and HLA-DR− were used as marker (CD44-APC: Molecular probes cat. n° A14749, Eugene, OR, USA; CD73-PE: cat. n° 55027, BD Biosciences Franklin Lakes, NJ, USA; CD29-PE: cat. n° 561795, BD Biosciences Franklin Lakes, USA; CD105-PerCP-Cy 5.5: cat. n°560819, BD Biosciences Franklin Lakes, USA; CD90-FITC: cat. n° 555595, BD Biosciences Franklin Lakes, USA; HLA-G-FITC: cat. n° MA1-19591, Invitrogen CA, USA; HLA-DR-FITC: cat. n° 555560, BD Biosciences Franklin Lakes, USA). The incubation was carried out in PBS + 0.5% bovine serum albumin for 30 min at room temperature, with the primary antibodies (dilution 1:50). Flow cytometry analysis was performed in a BD Accuri cytometer. Data were analyzed with FlowJo software.

Palma M.B., Luzzani C., Andrini L.B., Riccillo F., Buero G., Pelinski P., Inda A.M., Errecalde A.L., Miriuka S., Carosella E.D, & Garcia M.N. (2021). Wound Healing by Allogeneic Transplantation of Specific Subpopulation From Human Umbilical Cord Mesenchymal Stem Cells. Cell Transplantation, 30, 0963689721993774.

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Expanded PPCs Immunophenotyping by Flow

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For flow cytometry analysis of PPCs, the cells were harvested, washed with PBS, resuspended in PBS, and stained with fluorescent antibodies for 60 min at 4 °C in the dark. We used CD73-PE (eBioscience, San Diego, CA, USA), CD90-PE (eBioscience), CD105-PE (eBioscience), CD34-FITC (eBioscience), CD45-PE (eBioscience), and HLA-DR-FITC (eBioscience) to characterize the expanding PPCs. Isotype-matched antibodies served as controls.

Zou C., Lu Y., Teng X., Wang S., Sun X., Huang F., Shu G., Huang X., Guo H., Chen Z., Zhang J, & Zhang Y.A. (2017). MRI tracking of autologous pancreatic progenitor-derived insulin-producing cells in monkeys. Scientific Reports, 7, 2505.

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Phenotypic Characterization of CD11c+ Cells

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The cells were phenotypically characterized by flow cytometry using the following conjugated antibodies (Abs): mouse anti-human-HLA-ABC-FITC, HLA-DR-FITC, CD80-FITC, and CD11c-PE-Cy7 (eBioscience, San Diego, CA, USA). Briefly, cells were gently removed from the culture plates using cell scrapers. Then, the cells were centrifuged at 1000 rpm for 5 minutes at 4°C, washed with PBS, and incubated with Abs for 30 minutes. After being washed twice with PBS, samples were acquired on a FACSCalibur (BD Biosciences, Hershey, PA, USA) and analyzed using FlowJo software (Tree Star Inc., OR, USA). All the analyses were made in the CD11c⁺ cell population of each condition and sample.

González F.E., Chernobrovkin A., Pereda C., García-Salum T., Tittarelli A., López M.N., Salazar-Onfray F, & Zubarev R.A. (2018). Proteomic Identification of Heat Shock-Induced Danger Signals in a Melanoma Cell Lysate Used in Dendritic Cell-Based Cancer Immunotherapy. Journal of Immunology Research, 2018, 3982942.

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Multicolor Flow Cytometry Analysis

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The cells were stained with fluorescent dye–conjugated antibodies for 30 min on ice and washed twice with PBS. After resuspending in PBS containing 2% FBS, the cells were analyzed by fluorescence-activated cell sorting (FACS Calibur; BD Biosciences, Franklin, NJ, USA). The following antibodies were used in the analysis: CD34-FITC, CD73-FITC, CD90-FITC, CD90-PE, human leukocyte antigen (HLA)-ABC-FITC, HLA-DR-FITC (eBioscience, San Diego, CA, USA); CD44-FITC, CD105-APC (BD Biosciences); CD45-FITC (BioLegend, San Diego, CA, USA). In the case of fresh tissues, CD73-FITC, CD90-PE, and CD105-APC were used for triple staining. Antibodies against PD-L1-PE and PD-L2-APC were purchased from eBioscience.

Wang K.H., Chang Y.H., Harnod T, & Ding D.C. (2022). Endometrial Cancer-Infiltrating Mesenchymal Stem Cells Exhibit Immunosuppressive Effects. Cell Transplantation, 31, 09636897221104452.

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Phenotypic Characterization of Stem Cells

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The stem cells were dissociated with trypsin, washed with cold PBS, and then separately stained with immunoglobulin G (IgG) or the following monoclonal antibodies conjugated to PE, PerCP, APC, or FITC: Epcam-PE, CD44-FITC, CD29-FITC, CD49f-FITC, CD73-FITC, CD105-APC, CD90-FITC, CD34-PerCP, CD31-FITC, CD45-FITC, and HLA-DR-FITC (all from eBioscience, USA). Upon being washed with PBS, the labeled cells were resuspended, and at least 10⁵ events were acquired by using a BD Accuri™ C6 flow cytometer (BD, USA).

Wu B., Gao F., Lin J., Lu L., Xu H, & Xu G.T. (2021). Conditioned Medium of Human Amniotic Epithelial Cells Alleviates Experimental Allergic Conjunctivitis Mainly by IL-1ra and IL-10. Frontiers in Immunology, 12, 774601.

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Phenotyping Leukemic Cells from AML Patients

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Primary cells from peripheral blood of AML patients with hyperleukocytosis were obtained by leukapheresis before therapy initiation. The leukapheretic products were diluted 10-fold in phosphate buffered saline (PBS), and the mononuclear cell fraction was separated using Histopaque-1077 (Sigma, #H8889). The cells were resuspended in RPMI 1640 medium with 10% fetal calf serum and with antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin), and aliquots were used for analysis of surface markers by flow cytometry and for mRNA isolation.
The antibodies used were as follows: CD45-V450 (#560367), CD4-BUV395 (#564724), CD8-BUV395 (#563795), CD19-BUV737 (#564303), CD34-BV786 (#743534), and CD371-BB515 (#565926) from BD Biosciences; HLA-DR-FITC (#11-9952-42), TIM-3-APC (#17-3109-42), and PD-L2-APC (#17-5888-42) from eBioscience; CLIP-PE (sc-12725 PE) from Santa Cruz; PD-L1-PE (#1P-177-T100), CD47-FITC (#1F-225-T100), and CD38-PE (#1P-366-T100) from Exbio (Prague, Czech Republic). HLA class I antibody (Abcam, ab2217) was conjugated in house using the Lightning-Link Fluorescein Conjugation kit (#707-0010, Innova Biosciences).

Kuželová K., Brodská B., Marková J., Petráčková M., Schetelig J., Ransdorfová Š., Gašová Z, & Šálek C. (2022). NPM1 and DNMT3A mutations are associated with distinct blast immunophenotype in acute myeloid leukemia. Oncoimmunology, 11(1), 2073050.

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Comprehensive Flow Cytometry Analysis

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The following antibodies were used for flow cytometry analysis: CD11c APC, CD80 FITC, CD83 FITC, CD86 PE, HLA-DR FITC, CD1a FITC, CD14 FITC, CD4PE, CD4 PE-Cy7, CD45RA PE, CD45RO FITC, CD25 PE-Cy7, CTLA-4 APC, CD39 PerCP-eFluor710, FOXP3 Alexa Fluor 700, IL-10 PE, IFN-γ PE-Cy7, and IL-17 APC (all eBioscience). For surface staining, cells were incubated in PBS 10% FBS containing the respective antibodies for 30 min at 4°C, washed, and stored in IC fixation buffer until analysis (eBioscience). Intracellular FoxP3 was detected using FoxP3-staining kit (eBioscience) according to the manufacturer’s instructions. For intracellular cytokines detection, cells were treated with 50 ng/ml PMA, 1 μg/ml ionomycin, and 1 μl/ml brefeldin A for 5 h. After harvesting, cells were surface stained for CD4. Intracellular cytokine staining was performed in permeabilization buffer (eBioscience) before cells were washed and resuspended in FACS buffer for flow cytometry analysis. Cell viability was assessed by 7-AAD and annexin-V PE staining (eBioscience). Data were collected on FACSCalibur and FACSAria cytometers (Beckton Dickinson, San Diego, CA, USA) and analyzed with Weasel v3.0.2 software.

Maggi J., Schinnerling K., Pesce B., Hilkens C.M., Catalán D, & Aguillón J.C. (2016). Dexamethasone and Monophosphoryl Lipid A-Modulated Dendritic Cells Promote Antigen-Specific Tolerogenic Properties on Naive and Memory CD4+ T Cells. Frontiers in Immunology, 7, 359.

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Multicolor Flow Cytometry Immunophenotyping

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Cells were washed once with FACS buffer (PBS−/−, 1% BSA, 2 mM EDTA, 0.1% NaN₃) and after FcR blocking (Human TruStain FcX, Biolegend) co-stained for: HLA-DR-FITC (1:50, clone: L243), CD86-PE-Cy7 (1:20, clone: IT2.2), CD163-APC (1:20, clone: GHI/61); all from eBiosciences (Hertfordshire, UK) and CD206-PE (1:20, clone: 15-2), (Biolegend, London, UK). After 30 min incubation at 4°C in the dark, cells were washed twice with FACS buffer (1500 rpm, 4°C, 5 min) and re-suspended in 500 μl DAPI solution (5ug/ml, Biolegend) for viability and immediately acquired on a BD FACS CANTO II flow cytometer and analyzed using FlowJo 7.6.5 software (Tree Star). Purity of isolated CD14+ cells was checked by staining for CD14-PerCP-Cy5.5 (1:50, clone: HCD14, Biolegend) and was routinely > 95% across all the experiments.

Georgouli M., Herraiz C., Crosas-Molist E., Fanshawe B., Maiques O., Perdrix A., Pandya P., Rodriguez-Hernandez I., Ilieva K.M., Cantelli G., Karagiannis P., Mele S., Lam H., Josephs D.H., Matias-Guiu X., Marti R.M., Nestle F.O., Orgaz J.L., Malanchi I., Fruhwirth G.O., Karagiannis S.N, & Sanz-Moreno V. (2019). Regional Activation of Myosin II in Cancer Cells Drives Tumor Progression via a Secretory Cross-Talk with the Immune Microenvironment. Cell, 176(4), 757-774.e23.

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Multiparameter Flow Cytometry Analysis

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Antibodies used for analysis were anti-human CD11c APC, CD80 FITC, CD83 FITC, HLA-DR FITC, CD40 PE, CD86 PE, TLR-2 PE, CXCR4 PE, CCR7 PE, CD4 PECy7, and IFN-γ APC (all from eBioscience). Cells were resuspended in PBS supplemented with fetal bovine serum (FBS) (HyClone Thermo Scientific, Logan, UT, USA), stained with specific antibodies, fixed with IC fixation buffer (eBioscience) and resuspended in FACSFlow buffer (Becton Dickinson, San Diego, CA, USA) for subsequent analysis. Data were acquired on a FACSAria III with FACSDiva v6.1.3 software (both Becton Dickinson) and analyzed by FlowJo software (Treestar, USA).

García-González P.A., Schinnerling K., Sepúlveda-Gutiérrez A., Maggi J., Hoyos L., Morales R.A., Mehdi A.M., Nel H.J., Soto L., Pesce B., Molina M.C., Cuchacovich M., Larrondo M.L., Neira Ó., Catalán D.F., Hilkens C.M., Thomas R., Verdugo R.A, & Aguillón J.C. (2016). Treatment with Dexamethasone and Monophosphoryl Lipid A Removes Disease-Associated Transcriptional Signatures in Monocyte-Derived Dendritic Cells from Rheumatoid Arthritis Patients and Confers Tolerogenic Features. Frontiers in Immunology, 7, 458.

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