Cd86 percp cy5

Manufactured by BD

The CD86-PerCP-Cy5.5 is a fluorescently-labeled monoclonal antibody that binds to the CD86 surface antigen. CD86 is a co-stimulatory molecule expressed on antigen-presenting cells, and is involved in T cell activation. The PerCP-Cy5.5 fluorescent label allows for detection of CD86-positive cells using flow cytometry.

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5 protocols using cd86 percp cy5

Monocyte-Derived Dendritic Cell Protocol

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Monocytes were isolated using human enrichment cocktail (STEMCELL Technologies, Grenoble, France) and thereafter were cultured in Roswell Park Memorial Institute (RPMI) medium containing 50 ng/ml each of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 (ImmunoTools, Friesoythe, Germany). After 72 h of incubation, half of the medium was replaced with fresh medium. After 6 days of culture, cells were collected and 2 × 10⁶/ml were cultured in the presence or absence of 10 μg/ml MDA-HSA prepared as described previously (18) . Endotoxin was not detected in MDA and HSA by the Limulus Amebocyte Lysate test.
The cells collected after 24 h were ≥90% viable. DCs were stained with antibodies against CD11C-PE/BV421, CD86-Percp-Cy5.5, CD80-FITC, and CD40-FITC (BD Bioscience, San Jose, California).

Rahman M., Steuer J., Gillgren P., Végvári Á., Liu A, & Frostegård J. (2019). Malondialdehyde Conjugated With Albumin Induces Pro-Inflammatory Activation of T Cells Isolated From Human Atherosclerotic Plaques Both Directly and Via Dendritic Cell–Mediated Mechanism. JACC: Basic to Translational Science, 4(4), 480-494.

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Monocyte-Derived Dendritic Cell Activation by HSPs

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Monocytes were isolated by human monocyte enrichment cocktail (Stemcell Technologies, France). Isolated monocytes were cultured with recombinant human granulocyte macrophage colony‐stimulating factor and interleukin‐4 (IL‐4) (Immunotools, Germany), 50 ng/mL of each in RPMI complete media. At day 3, half of the media was replaced with fresh RPMI complete media with granulocyte macrophage colony‐stimulating factor and IL‐4. At day 6, 2×10⁶/mL cells were cultured with or without HSP60 (MyBiosource) or HSP90 (HSPß90, Sigma Aldrich, MO) at the concentrations described. Cells were collected after 24 hours and analyzed with >90% cell viability. DC stained with CD11C‐PE, CD86‐Percp/Cy5.5, CD83‐APC, CD40‐fluorescein isothiocyanate (BD Bioscience), and HLA‐II‐fluorescein isothiocyanate (Biolegend) antibodies.
The endotoxin concentration in HSP60 was checked by LAL Chromogenic Endotoxin Quantitation Kit (Thermofisher Scientific, IL). The level of endotoxin in the HSP60 was ≤1 EU/mg, according to manufacturer information, which is equal to ≤0.1 ng/mg. Our working concentration 5 μg/mL of HSP60 contains ≤0.0005 ng endotoxin. A similar concentration of endotoxin was determined in HSP90.

Rahman M., Steuer J., Gillgren P., Hayderi A., Liu A, & Frostegård J. (2017). Induction of Dendritic Cell–Mediated Activation of T Cells From Atherosclerotic Plaques by Human Heat Shock Protein 60. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 6(11), e006778.

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Multiparameter Flow Cytometry Staining

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Cells were stained for 20 min on ice in MACS buffer (2% FCS in PBS with 1 mM EDTA) at 0.5 to 1 × 10⁶ cells per well in 96-well round-bottom plates unless otherwise specified. All mAbs were purchased from BioLegend unless otherwise indicated. The following monoclonal antibodies were used: TCRβ–BV421, TCRβ–PerCP-Cy5.5 (Tonbo), CD23–PE-Cy7, CD23–Alexa Fluor 488, CD86–Alexa Fluor 647, CD86–PerCP-Cy5.5, CD21/35–Pacific Blue, B220–BV785, CD69–FITC (BD), CD93–APC (eBioscience), CD4–BV605, CD45.2–PE, CD45.1–BV605, lambda-1 light chain–biotin (BD) (followed by streptavidin–BV605), CD19–PE, and CCR7–biotin (followed by streptavidin–BV421). CCR7-biotin was stained for 1 h at room temperature followed by streptavidin and other surface markers on ice. Dead cells were excluded using Fixable Viability Dye eFluor780 (eBioscience no. 65-0865-18). For time-course experiments all samples were plated simultaneously, collected and kept on ice, and stained in parallel. Viability for 24- to 48-h cultures was determined by pregating on B220⁺ TCRβ^--. All samples were run on a BD LSRii at 5,000 to 10,000 events per second. Flow cytometry data were analyzed using FlowJo (v10.8.0).

Wolf E.W., Howard Z.P., Duan L., Tam H., Xu Y, & Cyster J.G. (2022). GPR174 signals via Gαs to control a CD86-containing gene expression program in B cells. Proceedings of the National Academy of Sciences of the United States of America, 119(23), e2201794119.

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Multi-parameter Flow Cytometry Analysis

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Multi-parameter flow cytometric analysis (FCM) was performed on a daily basis using the same antibody panel from the cell sort, without 7AAD and including CD69-APC-Cy7, CD86-PerCP-Cy5.5 and CD134-PE-Cy5 (BD Biosciences), to assess the phenotype and cellular activation status of each cell type in whole blood, the PBMC and PMN fractions, and post-sorted cell samples as previously described (see S2 Text for additional details) [29 (link)].

Howard L.M., Hoek K.L., Goll J.B., Samir P., Galassie A., Allos T.M., Niu X., Gordy L.E., Creech C.B., Prasad N., Jensen T.L., Hill H., Levy S.E., Joyce S., Link A.J, & Edwards K.M. (2017). Cell-Based Systems Biology Analysis of Human AS03-Adjuvanted H5N1 Avian Influenza Vaccine Responses: A Phase I Randomized Controlled Trial. PLoS ONE, 12(1), e0167488.

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Phenotypic Analysis of Cryopreserved PBMCs

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Cryopreserved PBMCs were thawed at 37°C, washed in RPMI/60% and RPMI/20% of fetal bovine serum (FBS), and incubated for 1 h at 37°C in RPMI/10%FBS. PBMCs were then stained with the Fixable Viability Stain-FVS780r (APC-H7 detect, BD Biosciences) for 15 mins. After PBMCs washing in PBS/1%FBS, cells were incubated in a U-bottom 96-well plate at a density of 1.5 million/well and stained with selected 14-color panel including: anti-CD19-AF700 (Clone HIB19), anti-IgD-Pe-Cy7 (Clone IA6-2), anti-IgM-BB515 (Clone G-20-127), polyclonal goat anti-IgA-Dylight^®649 (from Jackson Immunoresearch), CD10-BV650 (Clone H10a), CD21-PE-CF594 (Clone B-LY4), CD27-BV510 (Clone L128), CD38-BV786 (Clone HIT2), CD45-RB-PE (Clone MT4), CD86-PerCP-Cy5.5 (Clone 2331), CD279 (PD-1)-BV421 (Clone EH12.1), all from BD Biosciences unless indicated, for 15 mins. After washing twice in PBS/1% FBS, cells were fixed in PBS/1% formaldehyde, acquired in a BD LSRFortessa (BD Biosciences) cytometer using a plate HTS loader (BD Biosciences) and analyzed with FlowJo software (Tree Star). Gating strategy is described in Supplementary Figure 1. Lymphocyte gate was defined manually by morphological parameters excluding non-viable cells. B cells were identified as CD19+ CD21+/− cells and gated according to the expression of different markers to identify B-cell maturation stages as described in Supplementary Figure 1.

Jiménez M., Pastor L., Urrea V., Rodríguez de la Concepción M.L., Parker E., Fuente-Soro L., Jairoce C., Mandomando I., Carrillo J., Naniche D, & Blanco J. (2021). A Longitudinal Analysis Reveals Early Activation and Late Alterations in B Cells During Primary HIV Infection in Mozambican Adults. Frontiers in Immunology, 11, 614319.

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