Peripheral blood mononuclear cells (PBMCs) were stained in four panels containing anti-CD3-PacificBlue (PacBlue) (antibodies from BD Biosciences unless otherwise indicated), anti-CD3-Alexa700, anti-CD25-PE-Cy7, anti-CD38-PE, anti-HLA-DR-PE-Cy7, anti-CCR5-APC, anti-CD123-PerCP-Cy5.5, anti-CD16-PacBlue, anti-CD80-FITC, anti-CD83-PE, anti-CD86-APC, anti-PD1-FITC, anti-PD-L1-PE, anti-HLA class I-APC, anti-CD69-APC-Cy7; anti-CD4-Qdot655, anti-CD8-PE-Cy5.5, anti-CD14-Qdot605 (Invitrogen); anti-CD45RA-ECD, anti-CD127-PE, anti-HLA-DR-ECD, anti-CD20-ECD (Beckman Coulter); anti-CD11c-Alexa700 (eBioscience); and anti-CD27-APC-Cy7 (BioLegend). A staining reagent for dead cells (Invitrogen Aqua Live/Dead Fixable Stain) was included. Cells were then washed and fixed in PBS containing 1% paraformaldehyde or permeabilized using a FOXP3 Fix/Perm kit (BioLegend) according to the manufacturer's instructions, intracellularly stained with anti-Ki67-Alexa 488 (BD Biosciences), anti-FOXP3-PacBlue, anti-T-Bet-BV711 (BioLegend), and anti-Eomes-eFluor 660 (eBioscience), fixed, and analyzed with a LSR II cytometer (Becton Dickinson) and FlowJo.
Anti cd86 apc
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-CD86-APC is a fluorescently labeled antibody that binds to the CD86 antigen, also known as B7-2. CD86 is a co-stimulatory molecule expressed on the surface of antigen-presenting cells, such as dendritic cells and B cells, and plays a role in the activation of T cells. The APC (allophycocyanin) fluorescent label allows for the detection and analysis of cells expressing CD86 using flow cytometry.
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Lab products found in correlation
Anti cd80 pe, by Thermo Fisher Scientific (4 mentions)
Anti cd83 pe, by Thermo Fisher Scientific (3 mentions)
Anti cd16 cd32 antibody, by BioLegend (2 mentions)
Anti mhcii apc, by Thermo Fisher Scientific (2 mentions)
Anti cd11c fitc apc, by Thermo Fisher Scientific (2 mentions)
Anti hamster igg fitc pe apc, by Thermo Fisher Scientific (2 mentions)
Anti rat igg2b apc, by Immunotools (2 mentions)
Anti cd40 pe, by Thermo Fisher Scientific (2 mentions)
Anti rat igg2a pe, by Thermo Fisher Scientific (2 mentions)
Cellquest pro software, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Lsrii cytometer, by BD (1 mentions)
Anti pd 1 fitc, by Thermo Fisher Scientific (1 mentions)
Anti t bet bv711, by BioLegend (1 mentions)
Anti cd45ra ecd, by Beckman Coulter (1 mentions)
Anti cd27 apc cy7, by BioLegend (1 mentions)
Anti cd3 alexa 700, by BD (1 mentions)
Anti cd20 ecd, by Beckman Coulter (1 mentions)
Anti cd8 pe cy5, by Thermo Fisher Scientific (1 mentions)
Live dead fixable aqua stain, by Thermo Fisher Scientific (1 mentions)
9 protocols using anti cd86 apc
1
Comprehensive PBMC Immunophenotyping Protocol
2
Quantification of M2 Macrophages
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For this assay, 106 cells/well were incubated for 48 h with 100 μg/mL P2C7 or PBS as the negative control. After incubation, macrophages were washed with PBS and removed from the plate with cold PBS (plus 10 mM EDTA). The cells were stained with anti CD206—FITC (Invitrogen, MR5D3), anti-F4/80—PE-Cy5 (eBioscience, BM8), anti-CD80-PE (eBioscience, 16-10A1), anti-F4/80—FITC (BM8), anti-MHC II—APC-Cy7 (Invitrogen, M5/114.15.2) and anti-CD86—APC (Invitrogen, GL1) in PBS, 4% FBS for 30 min. Further labeled cells were analyzed in a flow cytometer (FACS Canto, BD Biosciences, San Diego, CA, USA). FACS characterization of M2 macrophages is shown in the Supplementary Materials (C. Macrophage characterization; Figure S8) .
3
Isolation and Characterization of Immune Cells from Mouse Spinal Cord
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The spinal cord, dissected from mice transcardially perfused with PBS, was minced into 1 mm3 pieces in solution containing 1 mg/mL or 0.1 mg/mL collagenase IV (Worthington Biochemical Corporation, USA) plus 0.4 mg/mL DNase I (Roche, Switzerland), and incubated at 37 °C for 15 min. For isolation of immune cells and microglia, cells were resuspended in 37% Percoll (GE Healthcare, USA) and centrifuged at 780 × g for 20 min. After centrifugation, myelin debris was removed and the cell pellet was collected. Cells were incubated with anti-CD16/CD32 antibodies (eBioscience, USA) for blocking Fc receptors, and then stained with combinations of the following antibodies: anti-CD45-PE-Cy7, anti-CD45-APC-Cy7, anti-CD4-PE, anti-CD8a-FITC, anti-CD8a-APC-Cy7, anti-NK1.1-PErCP-Cy5.5, anti-NK1.1-PE, anti-CD11c-PE, anti-CD11b-APC-Cy7, anti-CD11b-PerCP-Cy5.5 (BD Biosciences, USA), anti-CD4-APC, anti-CD3e-PerCP-Cy5.5, anti-CD86-APC, anti-CD25-PE (eBioscience, USA), anti-CD11c-APC, anti-I-A/I-E (MHC class II)-FITC, anti-CD4-PE-Cy7, anti-CD4-APC, anti-CD68-PE, anti-H-2Kb/H-2Db (MHC class I)-PE, anti-CD206-PE, anti-CD178 (FasL)-PE, and Armenian Hamster IgG Isotype control-PE (BioLegend, USA). Flow cytometry was performed by using FACS Aria and FACS Verse flow cytometers (BD Biosciences, USA) and the data was further analyzed using FlowJo Software (FlowJo, LLC, USA).
4
Flow Cytometry Analysis of Bone Marrow-Derived Dendritic Cells
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Flow cytometry of BMDCs was performed as described in Yin et al (2015) (link), but with some modifications. In brief, 5 × 105 BMDCs were preincubated for 20 min on ice with supernatant collected from hybridoma clone 2.4G2 cell culture (Unkeless, 1979 (link)) to block non-specific binding of Fc γ receptor, followed by staining for 20 min on ice with a fluorescent antibody cocktail containing anti-CD11c-PE (BioLegend), anti-CD11b-APC-eFluor 780 (eBioscience), anti-CD86-APC (eBioscience), and anti-MHC class II–PB (BioLegend). After washing three times with FACS buffer (PBS containing 2% FBS) and resuspension in FACS buffer, the BMDCs were analyzed by flow cytometry (BD LSR-II; BD Biosciences).
5
Analyzing Dendritic Cell Responses to Mannosylated Carbon Nanotubes
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To detect intracellular OVA signals, FITC‐OVA, MWCNTs‐COOH/FITC‐OVA or Man‐MWCNTs/FITC‐OVA was incubated with BMDCs for 4 h. After washing the BMDCs with FACS buffer (phosphate buffer saline (PBS) containing 1 % FBS), the cellular fluorescence was measured by FCM analysis.
To measure the effects of Man‐MWCNTs/OVA on the maturation of BMDCs, BMDCs were also treated with OVA, Man‐MWCNTs or Man‐MWCNTs/OVA for 12 h. Then cells were washed with FACS buffer and subsequently stained with anti‐CD11c‐PE−Cy7, anti‐CD86‐APC, and anti‐CD80‐PE antibodies (eBioscience) for 30 min at 4 °C. These BMDCs were then sorted by flow cytometry after being washed again with FACS buffer. All samples were done in triplicate.
To measure the effects of Man‐MWCNTs/OVA on the maturation of BMDCs, BMDCs were also treated with OVA, Man‐MWCNTs or Man‐MWCNTs/OVA for 12 h. Then cells were washed with FACS buffer and subsequently stained with anti‐CD11c‐PE−Cy7, anti‐CD86‐APC, and anti‐CD80‐PE antibodies (eBioscience) for 30 min at 4 °C. These BMDCs were then sorted by flow cytometry after being washed again with FACS buffer. All samples were done in triplicate.
6
T cell Receptor Imaging and Activation
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The reagents used in our experiments included: OVA(323–339) (Sigma-Aldrich, St Louis, MO, USA), thapsigargin (TG, stock 1 mM in DMSO, Molecular Probes, Invitrogen, USA), cytochalasin D and nocodazole (Calbiochem, Merck KGaA, Germany). Recombinant murine interferon gamma (IFN-γ) was purchased from Peprotech (Rocky Hill, NJ, USA). For live cell imaging, H57-597-Fab-TCRαβ-Alexa Fluor 647 was purchased from Invitrogen to label TCR as a non-blocking antibody [22 (link)]. For calcium imaging, Calcium Crimson™ was purchased from Invitrogen. The following antibodies (Abs) were used for immunofluorescence: Texas Red-X phalloidin for anti-F-actin, MitoTracker® Green FM (Molecular Probes, Invitrogen, USA), anti-PLCγ1 (sc81, Santa Cruz Biotechnology, Santa Cruz, CA, USA), polyclonal rabbit anti-PKC-θ (sc212, Santa Cruz Biotechnology), anti-ZAP-70 (Y319, Abcam, Cambridge, MA, USA), anti-ORAI1 (ab59330, Abcam, Cambridge, MA, USA) and anti-PMCA (5 F10, Abcam, Cambridge, MA, USA), Dylight 405-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories), Alexa Fluor 555-conjugated goat anti-rabbit IgG (Invitrogen). The following Abs were used for flow cytometry: anti-MHC-II (I-Ab)-FITC, antiCD80-PE, anti-CD86-APC and anti-CCR7-APC (all from eBioscience).
7
Phenotypic Analysis of LPS-Stimulated cDCs
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On day 10, LPS-stimulated cDCs and unstimulated cells were harvested by centrifugation and resuspended in PBS. An average of 5 × 105 cells per stain was subjected to subsequent analyses. Prior to staining, Fc receptors on cDCs were blocked by preincubation with anti-CD16/CD32 antibodies (Biolegend) for 5-10 min on ice. Surface staining was performed by incubating the cells with fluorochrome-conjugated specific antibodies, or the corresponding isotype controls, for at least 20 min in dark on ice. The following antibodies and isotype controls (unless stated otherwise, all purchased from Miltenyi Biotec, Bergisch Gladbach, Germany) were employed: anti-CD11c-FITC/-APC (#130-102-466/-493), anti-MHC-II-APC (#130-102-139), anti-CD40-PE (#130-102-599), anti-CD80-PE (#130-102-613), anti-CD83-PE (#130-104-474), anti-CD86-APC (#130-102-558), anti-hamster IgG-FITC/-PE/-APC (eBiosciences, San Diego, CA, USA), anti-rat IgG2a-PE (eBiosciences), anti-rat IgG2b-APC (Immunotools), and REA Control. Flow cytometric analyses were performed on a FACSCalibur (BD Biosciences). A total of 20,000 events per sample were acquired, and data were evaluated using the CellQuestPro software (BD Biosciences).
8
Characterization of DC Maturation and T-Cell Subsets
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The maturation of DCs was examined by staining with fluorescence‐conjugated antibodies including anti‐CD11c‐FITC (eBioscience, catalog: 11‐0114‐85), anti‐CD86‐APC (eBioscience, catalog: 17‐0862‐82), and anti‐MHC Class II‐PE (eBioscience, catalog: 12‐5321‐82). To analyze the T‐cell subsets in spleens and draining lymph nodes of the immunized mice, single cell suspensions prepared from these samples were examined by flow cytometry. The following primary antibodies were used: anti‐CD3e‐PerCP‐Cyanine5.5 (eBioscience, catalog: 45‐0031‐82), anti‐CD8a‐PE (eBioscience, catalog: 12‐0081‐82), anti‐CD4‐FITC (eBioscience, catalog: 11‐0041‐85), anti‐CD25‐APC (eBioscience, catalog: 17‐0251‐82), anti‐Foxp3‐PE (eBioscience, catalog: 12‐4771‐82), anti‐IFN‐γ‐FITC (BD Bioscience, catalog: 554 411), anti‐TNF‐α‐Alexa Fluor 647 (BD Bioscience, catalog: 557 730), and anti‐CD16/CD32 (eBioscience, catalog: MA5‐18012). Flow data were acquired on a CytoFLEX and analyzed using CytExpert (Beckman Coulter, USA) and FlowJo (TreeStar, USA) softwares.
9
Flow Cytometric Analysis of Activated cDCs
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