Hla dr percp l243

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HLA-DR-PerCP (L243) is a flow cytometry reagent used for the detection and quantification of HLA-DR expression on the surface of cells. HLA-DR is a major histocompatibility complex (MHC) class II cell surface receptor that plays a crucial role in the presentation of peptide antigens to T cells. The L243 clone is conjugated to the PerCP (Peridinin Chlorophyll Protein Complex) fluorescent dye, allowing for the visualization and analysis of HLA-DR-positive cells.

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6 protocols using hla dr percp l243

Plasmacytoid and Myeloid Dendritic Cell Analysis

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PBMCs were isolated from whole blood according to a previous report [23 (link)]. PBMCs were stained with fluorescent dye conjugated to antibodies. For pDC, anti-Human CD123-FITC (AC145) (Miltenyi Biotec., Bergisch Gladbach, Germany), BDCA4-APC (AD-17F6) (Miltenyi Biotec.), CD86-PE (IT2.2) (eBioscience, San Diego, CA, USA), and HLA-DR-PerCP (L243) (BD Bioscience, NJ, USA) were used. For mDC, anti-human Lineage Cocktail 1-FITC (Lin1) (CD3, CD14, CD16, CD19, CD20, CD56) (MφP9, NCAM16.2, 3G8, L27, SJ25C1, SK7) (BD Bioscience), CD11c-APC (MJ4-27G12) (Miltenyi Biotec.), CD1c-PE-Cy7 (L161) (BioLegend, MS, USA), CD86-PE (IT2.2) (eBioscience), and HLA-DR-PerCP (L243) (BD Bioscience) were used. CD123⁺BDCA4⁺ cells were defined as pDC and Lin1⁻CD1c⁺CD11c⁺ cells were identified as mDC. The expression levels of HLA-DR and CD86 were used as maturation markers of pDC and mDC. After staining, cells were analyzed by flow cytometry using FACS Cant II (BD Biosciences). Data were analyzed using the FlowJo software (Treestar, ON, USA).

Komano Y., Shimada K., Naito H., Fukao K., Ishihara Y., Fujii T., Kokubo T, & Daida H. (2018). Efficacy of heat-killed Lactococcus lactis JCM 5805 on immunity and fatigue during consecutive high intensity exercise in male athletes: a randomized, placebo-controlled, double-blinded trial. Journal of the International Society of Sports Nutrition, 15, 39.

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Evaluating pDC and mDC activation

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Peripheral blood mononuclear cells (PBMCs) were isolated from the blood [17 (link)]. To evaluate pDC activity, the PBMCs were incubated with anti-human CD123-FITC (AC145) (Miltenyi Biotec., Bergisch Gladbach, Germany), BDCA4-APC (AD-17F6) (Miltenyi Biotec.), CD86-PE (IT2.2) (eBioscience, San Diego, CA, USA), and HLA-DR-PerCP (L243) (BD Bioscience, Franklin Lakes, NJ, USA) and with anti-human Lineage Cocktail1-FITC (Lin1) (CD3, CD14, CD16, CD19, CD20, CD56) (MφP9, NCAM16.2, 3G8, L27, SJ25C1, SK7) (BD Bioscience), CD11c-APC (MJ4-27G12) (Miltenyi Biotec.), CD1c-PE-Cy7 (L161) (BioLegend, San Diego, CA, USA), CD86-PE (IT2.2) (eBioscience), and HLA-DR-PerCP (L243) (BD Bioscience) to evaluate mDC activity. CD123 positive + BDCA4 positive cells were defined as pDCs, and Lin1 negative + CD11c positive + CD1c positive cells were defined as mDCs. The expression intensities of the cell surface molecules CD86 and HLA-DR were used as a marker of pDC and mDC activation. Data were collected by flow cytometer (FACS Cant II, BD Biosciences), and FlowJo software (Treestar, Ashland, OR, USA) was used for data analysis.

Komano Y., Fukao K., Shimada K., Naito H., Ishihara Y., Fujii T., Kokubo T, & Daida H. (2023). Effects of Ingesting Food Containing Heat-Killed Lactococcus lactis Strain Plasma on Fatigue and Immune-Related Indices after High Training Load: A Randomized, Double-Blind, Placebo-Controlled, and Parallel-Group Study. Nutrients, 15(7), 1754.

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Longitudinal Immune Profiling in Humanized Mice

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Immunophenotyping was performed on peripheral blood samples collected longitudinally and at the study endpoint on whole blood and on mononuclear cells isolated from the tissues of humanized mice. Prior to antibody incubation, Ig-binding sites were blocked. The fluorochrome-conjugated antibodies included CD45-V500 (HI30, BD Biosciences, San Jose, USA), CD3-APC-R700 (UCHT1, BD Biosciences, San Jose, USA), CD4-APC-H7 (RPA-T4, BD Biosciences, San Jose, USA), CD8-FITC (SK1, BD Biosciences, San Jose, USA), CD19-PE-Cy7 (SJ25C1, BD Biosciences, San Jose, USA), CD27-PE (M-T271, BD Biosciences, San Jose, USA), CD38-APC (HB7, BD Biosciences, San Jose, USA), CD45RA-Pacific Blue (F8-11-13, Bio-Rad, Hercules, USA), HLA-DR-PerCP (L243, BD Biosciences, San Jose, USA), mouse IgG1k-APC (MOPC-21, BD Biosciences, San Jose, USA), mouse IgG1k- Pacific Blue (MOPC-21, BD Biosciences, San Jose, USA), mouse IgG1k-PE (MOPC-21, BD Biosciences, San Jose, USA) and mouse IgG2ak-PerCP (X39, BD Biosciences, San Jose, USA). After antibody incubation, blood samples were lysed with t 1× BD FACS lysing solution (BD Biosciences, San Jose, USA). Samples were then analyzed on a BD LSRFortessa instrument (BD Biosciences, San Jose, USA).

Ling L., Leda A.R., Begum N., Spagnuolo R.A., Wahl A., Garcia J.V, & Valente S.T. (2023). Loss of In Vivo Replication Fitness of HIV-1 Variants Resistant to the Tat Inhibitor, dCA. Viruses, 15(4), 950.

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Flow Cytometry Analysis of T Cell Subsets

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Whole blood was stained for flow cytometry with multiple combinations of the following mAbs: CD3-FITC (SP34) ; CD20-PE (3G8); CD8-PerCP (SK1); CD4-APC (L200); CD25-FITC (2A3); HLA-DR-PerCP (l243); Ki-67-FITC (B56) (all from BD Biosciences) and FoxP3-APC (PCH101) (EBiosciences), as described (17 (link), 18 (link)). Data were acquired with a FACSCalibur flow cytometer (BD Immunocytometry Systems) and analyzed with CellQuest software (BD Biosciences). CD4⁺ and CD8⁺ T cell percentages were obtained by first gating on lymphocytes and then on CD3⁺ T cells. Immune activation, and proliferation markers were determined by gating on lymphocytes, then on CD3⁺ T cells, and finally on CD4⁺CD3⁺ or CD8⁺CD3⁺ T cells.

He T., Brocca-Cofano E., Policicchio B.B., Sivanandham R., Gautam R., Raehtz K.D., Xu C., Pandrea I, & Apetrei C. (2016). T Regulatory Cell Depletion Reactivates Latent SIV in Controller Macaques while Boosting SIV-specific T Lymphocytes. Journal of immunology (Baltimore, Md. : 1950), 197(12), 4535-4539.

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Multicolor Flow Cytometry of Immune Cell Activation

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EDTA anticoagulated peripheral blood cells were labeled following a lyse/wash protocol with an 8-color/9-monoclonal antibody (mAb) panel including CD3 AmCyam (clone SK7, BD Biosciences), CD4 PECy7 (SK3, BD), CD8 APCCy7 (SK1, BD), CD16 PacBlue (3G8, BD), CD19 PECy7 (SJ25C1, BD), CD28 FICT (CD28.2, BD), CD38 APC (HB7, BD), CD86 PE (IT2.2, BD), and HLA-DR PerCP (L243, BD). Five microliters of each antibody in 100 µL of whole blood was incubated for 15 minutes at room temperature in the dark. Samples were lysed with 3 mL of 1X FACSlysing solution (BD) for 5 minutes and washed with 3 mL of FACSFlow (BD). Half a million cells were immediately acquired in a FACSCanto flow cytometer (BD), daily calibrated using 7-color setup beads (BD), and analyzed with DIVA software (BD) following the gating strategy described in Supplementary Figure 1.
The expression of CD28, CD38, CD86, and HLA-DR activation/senescence markers was evaluated as a percentage or absolute number (cells/µL) of positive cells as well as mean fluorescence intensity (MFI) of the marker on CD3⁺CD4⁺, CD3⁺CD8⁺, and CD3⁺CD4⁺CD8⁺ T lymphocytes, CD19⁺ B lymphocytes, CD3^-CD19^-CD16⁺ natural killer (NK) lymphocytes, monocytes (CD4⁺CD86⁺HLA-DR⁺ medium side scatter [SSC] cells), granulocytes (CD16⁺⁺ elevated SSC cells), and eosinophils (elevated SSC auto fluorescent cells).

Bernal E., Martinez M., Campillo J.A., Puche G., Baguena C., Tomás C., Jimeno A., Alcaraz M.J., Alcaraz A., Muñoz A., Oliver E., de la Torre A., Marín I., Cano A, & Minguela A. (2021). Moderate to Intense Physical Activity Is Associated With Improved Clinical, CD4/CD8 Ratio, and Immune Activation Status in HIV-Infected Patients on ART. Open Forum Infectious Diseases, 9(3), ofab654.

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Quantitative Analysis of Plasmacytoid Dendritic Cells

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PBMC were stained with fluorescent dye conjugated to Abs: CD123-FITC (AC145), BDCA4-APC (AD-17F6) (Miltenyi Biotec), CD86-PE (IT2.2) (eBioscience) and HLA-DR-PerCP (L243) (BD Pharmingen). After staining, cells were washed twice with fluorescence-activated cell sorting (FACS) buffer (0•5 % bovine serum albumin in PBS buffer) and suspended in 4 % paraformaldehyde for FACS analysis. Data were collected by FACS Cant II (BD Biosciences) and analysed by FCS Express software (De Novo Software). pDC were defined as CD123 + BDCA4 + , and the activation markers on pDC were measured. To eliminate the influence of analytical error, volunteers whose data were outliers (mean ± 2 SD) were excluded from the analysis.

Sugimura T., Takahashi H., Jounai K., Ohshio K., Kanayama M., Tazumi K., Tanihata Y., Miura Y., Fujiwara D, & Yamamoto N. (2015). Effects of oral intake of plasmacytoid dendritic cells-stimulative lactic acid bacterial strain on pathogenesis of influenza-like illness and immunological response to influenza virus. The British journal of nutrition, 114(5).

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