Cd86 pe it2

Manufactured by BD

The CD86 PE (IT2.2) is a fluorescently-labeled monoclonal antibody used for the identification and enumeration of CD86-expressing cells in flow cytometry applications. The PE (phycoerythrin) fluorescent label allows for the detection and analysis of CD86-positive cells.

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3 protocols using cd86 pe it2

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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Multiparameter Immunophenotyping of T-Cells

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To determine CD86, CD83 and HLA-DR on DCs the cells were washed twice using FACS buffer and were subsequently stained using CD86-PE (IT2.2, BD Biosciences), CD83-APC (HB15e, Biolegend) and HLA-DR-PerCp (L243, BD Biosciences) for 30min at 4°C in FACS buffer. Cells were washed once more with FACS buffer and were analysed on a FACS Canto II apparatus. To assess the capacity of T-cells to produce cytokines/ chemokines, cells were permeabilized with PermWash (BD Pharmingen) according to manufacturer’s protocol to stain for intracellular markers. Cells were stained for p24-RD1 (Beckman Coulter, KC57-RD1) and IFN-γ-FitC (4S.B3), IL2-PerCp-Cy5.5 (MQ1-17H12), IL-4-APC (MP4-25D2), TNF-α-PE-CF594 (MAb11), Mip-1β-AlexaFluor700 (D21-1351) (all from BD Bioscience) for 30min at 4°C in PermWash. Next, these cells were washed once with PermWash, resuspended in FACS buffer (PBS+ 2%FCS) and measured on a FACS Canto II apparatus. The histograms depict all T-cells capable of producing a certain cytokine/ chemokine at the optimal time point. The optimal time point was determined by the percentage of live cells (>50%) and the level of HIV-1 infection, which varied per donor and per virus between day 5 and 7.

Mouser E.E., Pollakis G., Yazdanbakhsh M., Harnett W., de Jong E.C, & Paxton W.A. (2016). Brugia malayi Antigen (BmA) Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells. PLoS ONE, 11(1), e0146527.

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Flow Cytometric Analysis of Immune Cell Markers

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For flow cytometric analysis, cells were washed with ice-cold FACS buffer (PBS supplemented with 3% i.a. FBS) and stained in 50 μl FACS buffer containing appropriate amounts of fluorescent-labelled antibodies. As a control for nonspecific binding, one aliquot of cells was labelled with isotype-specific control antibodies in excess concentration. After 30 minutes of incubation and extensive washing, the median fluorescence intensity (MFI) of 1 × 10⁴ cells was recorded for every sample using a FACS Canto II flow cytometer and FACS Diva software (both from BD Biosciences, Vienna, Austria). Anti-human CD14-FITC (clone: MEM-15), mouse IgG1-FITC (PPV-06), mouse IgG2a-APC (PPV-04) and mouse IgG2b-PE (PLRV219) were purchased from ImmunoTools (Friesoythe, Germany). Anti-human CD40-APC (5C3), CD80-PE (L307.4), CD83-APC (HB15e) and CD86-PE (IT2.2) were acquired from BD Biosciences, Vienna, Austria. Mouse IgG1-APC isotype control (11711) was purchased from R&D Systems (Biomedica, Vienna, Austria).

Schwarz H., Gornicec J., Neuper T., Parigiani M.A., Wallner M., Duschl A, & Horejs-Hoeck J. (2017). Biological Activity of Masked Endotoxin. Scientific Reports, 7, 44750.

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