Anti hladr v500

Manufactured by BD

Sourced in United States

The Anti-HLADR-V500 is a fluorochrome-conjugated monoclonal antibody that binds to the HLA-DR antigen, which is expressed on the surface of antigen-presenting cells. It is designed for use in flow cytometry applications to identify and analyze HLA-DR-positive cells.

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3 protocols using anti hladr v500

Isolation and Characterization of Plasmacytoid Dendritic Cells

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Fresh PBMCs were isolated from whole blood by density-gradient centrifugation using the BD Vacutainer CPT Mononuclear Cell Preparation Tubes with Sodium Heparin (BD Biosciences, Franklin Lakes, NJ, USA). The cells were first stained using the Zombie Yellow™ Fixable Viability Kit (BioLegend, San Diego, CA, USA) and then with combinations of the following monoclonal antibodies against human cell-surface antigens for 30 min on ice: anti-CD11c-Alexa700, anti-HLADR-V500, anti-CD19-APC-H7 (all from BD Biosciences), anti-CD14-ECD, anti-CD56-APC, (both from Beckman Coulter, Brea, CA, USA), anti-CD123-FITC, anti-CD3- PerCPCy5.5, anti-CD56-BV421 (all from BioLegend), and anti-CD19-PE (TONBO Biosciences, San Diego, CA, USA). pDCs were identified as CD3^-CD19^-CD14^-CD56^-HLADR⁺CD11c^-CD123⁺(Additional file 2: Figure S1). Data were acquired on a FACS LSR Fortessa (BD Biosciences) and the percentages of each cell population and mean fluorescence intensity were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).

Murayama G., Furusawa N., Chiba A., Yamaji K., Tamura N, & Miyake S. (2017). Enhanced IFN-α production is associated with increased TLR7 retention in the lysosomes of palasmacytoid dendritic cells in systemic lupus erythematosus. Arthritis Research & Therapy, 19, 234.

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Comprehensive Immune Profile of PBMCs

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After resting 2-3h in complete medium at 37°C, thawed PBMC were stained with the following mAbs: anti-CD4-Alexa Fluor 700 (BioLegend Inc., San Diego, CA, USA), anti-CD8-PE-Texas Red (Invitrogen), anti-CD45RA-Qdot655, anti-CCR7-BV421, anti-HLA-DR-V500 (BD Biosciences), and anti-PD-1-BV711 (BioLegend). Cells were subsequently fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), stained with anti-Ki67-PerCP eFluor710 (eBioscences, San Diego, CA) and anti-perforin-FITC (Diaclone, Besançon, France), and acquired with a LSR II Fortessa flow cytometer (BD Biosciences). Analysis was performed with FlowJo software (FlowJo LLC, Ashland, OR); after gating on total memory CD4⁺ and CD8⁺ lymphocytes, the percentage of IL-7R^pos and IL-7R^neg cells was calculated, as well as the percentage of IL-7R^pos and IL-7R^neg CD4⁺ T cells expressing Ki67, HLA-DR, PD-1 and perforin.

Mele F., Fornara C., Jarrossay D., Furione M., Arossa A., Spinillo A., Lanzavecchia A., Gerna G., Sallusto F, & Lilleri D. (2017). Phenotype and specificity of T cells in primary human cytomegalovirus infection during pregnancy: IL-7Rpos long-term memory phenotype is associated with protection from vertical transmission. PLoS ONE, 12(11), e0187731.

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Multiparametric Phenotyping of PBMCs

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PMBCs were thawed in Benzonase Nuclease (Sigma-Aldrich)/X-VIVO medium (Lonza), washed with RPMI medium and stained with the following fluorochrome-conjugated antibodies: anti-CD14 – PerCP-Cy5.5 (BD, clone MΦP9), anti-HLA-DR – V500 (BD, clone G46–6), anti-PD-L1 – PE-Cy7 (BD, clone MIH1), anti-CD73 – BV605 (Biolegend, clone AD2), anti-CD3 – V500 (BD, clone SP34–2), anti-CD4 – APC-Cy7 (BD, clone RPA-T4), anti-CD8 – APC (BD, clone RPA-T8), anti-CD69 – PE-Cy7 (BD, clone FN50), anti-PD-1 – PerCP-Cy5.5 (BD, clone EH12.1) and anti-CD25 – BV421 (BD, clone M-A251). Dead cells were discriminated with the Fixable Viability Stain A×700(BD). To reduce unspecific antibody binding, FcR Blocking Reagent (Miltany) was added. Analysis of the intracellular marker FOXP3 – Alexa 488 (BD, clone 259D/C7) were conducted using the FOXP3/Transcription Factor Fixation/Permeabilization kit (ThermoFisher). Acquisition was performed by 10-color flow cytometry using BD FACSLyric with FACSuite software (BD Biosciences). FlowJo V 10 software (BD Biosciences) was used to analyze at least 10⁶ events. Positively stained cells were gated according to the fluorescence minus one (FMO) control.

Lepper A., Bitsch R., Özbay Kurt F.G., Arkhypov I., Lasser S., Utikal J, & Umansky V. (2023). Melanoma patients with immune-related adverse events after immune checkpoint inhibitors are characterized by a distinct immunological phenotype of circulating T cells and M-MDSCs. Oncoimmunology, 12(1), 2247303.

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