DC maturation was analyzed 24 hours post stimulation. Cells were stained with PE-conjugated anti-CD80 (1:25, 557227, BD Pharmingen), APC-conjugated anti-CD83 (1:25, 551073, BD Pharmingen) and FITC-conjugated anti-CD86 (1:25; 555657; BD Pharmingen). Cells were analyzed on a FACS Canto II (BD Biosciences).
Fitc conjugated anti cd86
Manufactured by BD
Sourced in United States
FITC-conjugated anti-CD86 is a laboratory reagent that binds to the CD86 protein, also known as B7-2, expressed on the surface of antigen-presenting cells. It is conjugated with the fluorescent dye FITC (Fluorescein Isothiocyanate) for detection and analysis purposes.
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Lab products found in correlation
Pe conjugated anti cd80, by BD (4 mentions)
Pe conjugated anti cd11c, by BD (3 mentions)
Facscanto 2, by BD (2 mentions)
Fitc conjugated anti cd11c, by BD (2 mentions)
Apc conjugated anti cd83, by BD (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
D glucose, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti ccr1, by R&D Systems (1 mentions)
Pe conjugated anti ccr2, by R&D Systems (1 mentions)
D luciferin, by Promega (1 mentions)
Facscalibur, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Biotin conjugated anti b220, by BD (1 mentions)
Biotinylated peanut agglutinin pna, by Merck Group (1 mentions)
Percp conjugated streptavidin, by BD (1 mentions)
Fitc conjugated anti cd14, by BD (1 mentions)
Pe conjugated anti cd40, by BD (1 mentions)
Pe conjugated anti mhc 2, by BD (1 mentions)
11 protocols using fitc conjugated anti cd86
1
Dendritic Cell Maturation Analysis
2
Murine Dendritic Cell Differentiation
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RPMI 1640 was purchased from Life Technologies Corporation (Grand Island, NY, USA), containing 4.5 g/L D-Glucose, L-Glutamine and 110 mg/L Sodium Pyruvate. GM-CSF, IL-4 and TNF-α were purchased from Peprotech Asia (Revohot, Israel). Lipopolysaccharide (LPS) was purchased from Sigma Aldrich (Steinheim, Sweden). D-luciferin was purchased from Promega Corporation (Madison, WI, USA). The following antibodies were used for FACS analysis: fluorescein isothiocyanate (FITC)-conjugated anti-CD86, FITC-conjugated anti-I-A[b], FITC-conjugated anti-H-2Kb, Phycoerythrin (PE)-conjugated anti-CCR7, PE-conjugated anti-mouse CD40, PE-conjugated anti-CD11c and FITC conjugated anti-CD11c were purchased from BD PharMingen (SanDiego, CA). PE-conjugated anti-H-2Kb (SIINFEKL) was purchased from BioLegend (San Diego, CA). PE-conjugated anti-CCR1, PE-conjugated anti-CCR2, PE-conjugated anti-CXCR1 and PE-conjugated anti-CCR7 were purchased from R&D Systems (Minneapolis, MN, USA). All the isotype control antibodies were purchased from the same company as the detecting antibodies.
3
Phenotyping Immune Cell Subsets
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Erythrocyte-depleted spleen or bone marrow cells were stained and analyzed as previously described [31 (link)]. Half a million RBC-depleted splenocytes were incubated with mouse IgG (Sigma-Aldrich) for 15 min prior to staining with various combinations of directly-conjugated mAbs. The following directly conjugated mAbs were purchased from BD Biosciences (San Diego, CA): FITC-conjugated anti-CD86 (GL1), -CD95 (Jo2), -CD4 (RM4-5), -CD3 (145-2C11), -CD44 (IM7), -CD62L (MEL-14), -CXCR5 (2G8), and -GL7 (GL7); biotin-conjugated anti-B220 (RA3-6B2), -IgMa (DS-1), -CD24 (M1/69), -CD23 (B3B4), -CD138 (281–2), -CD19 (1D3) and -CD21 (7G6); allophycocyanin-conjugated anti-IFN-γ (XMG1.2); Alexa Fluor488-conjugated anti-IL17A (TC11-18H10); and PE-anti-IL21 (4A9). Biotinylated peanut agglutinin (PNA) was purchased from Sigma-Aldrich (St. Louis, MI) and biotinylated polyclonal rabbit anti-HEL Ab was purchased from Rockland (Gilbertsville, PA). Isotype controls were purchased from Caltag (Buckingham, UK). Allophycocyanin-, PE-, or PerCP-conjugated streptavidin (BD Biosciences) were used to reveal biotinylated Ab staining. Dead cells were excluded by staining with propidium iodide (PI, Sigma-Aldrich), 0.6 μg/ml. Stained cells were acquired and analyzed using a BD FACSCalibur and FACSDiva software, or using a BD LSRII flow cytometer and FlowJo software (TreeStar, San Carlos, CA).
4
Phenotypic Analysis of Dendritic Cells
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Phenotypic analysis was performed by direct immunofluorescence staining of DC cell surface markers. Cells (in PBS) were stained with the following antibodies at 4 °C for 20 min: Phycoerythrin (PE)-conjugated anti-CD11c (Clone N418); Fluorescein isothiocyanate (FITC)-conjugated anti-CD14 (Clone Sa14-2); PE-conjugated anti-CD40 (Clone 3/23); FITC-conjugated anti-CD54 (Clone 3E2); PE-conjugated anti-CD80 (Clone 16-10A1); FITC-conjugated anti-CD86 (Clone PO3); FITC-conjugated anti-MHC I (Clone KH95), or PE-conjugated anti-MHC II (Clone 2G9) (all antibodies were purchased from BD Pharmingen, San Diego, CA, USA), plus appropriate isotype controls. Cells were then analyzed using a 3-color FACSCalibur cytometer (BD Biosciences, Mountain View, CA). Data were collected from 10,000 events and analyzed using FlowJo10 software (BD Biosciences).
5
Cytokine Profiling of Monocytes
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Cell surface and intracellular cytokine staining of monocytes was performed as previously described (46 (link)). Briefly, cultured monocytes or purified monocytes were washed in PBS and stained by PE-conjugated anti-CD80 (BD Pharmingen, 557227), FITC-conjugated anti-CD86 (BD Pharmingen, 555657), PE-conjugated anti-CD163 (BD Pharmingen, 556018), FITC-conjugated CD206 (BD Pharmingen, 551135), PE-Cy7–conjugated HLA-DR (BD Pharmingen, 560651), or APC-conjugated anti-CD42b (BD Pharmingen, 551061) before analysis by FACSCanto. Fixation and permeabilization were performed using Cytofix/Cytoperm Plus kit with GolgiPlug (BD Biosciences) and stained by PE-conjugated anti–IL-1β (R&D Systems, IC201P) and PE-CF594–conjugated anti–IL-10 (BD Horizon, 562400) before analysis. All data acquired from flow cytometry were analyzed with FlowJo (Treestar software).
6
Flow Cytometry Analysis of Dendritic Cell Activation
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DCs were stimulated for 24 h and stained with PE-conjugated anti-CD80 (1:12.5, 557,227, BD pharmingen), allophycocyanin-conjugated CD83 (1:25, 551,073, BD Pharmingen), FITC-conjugated anti-CD86 (1,25, 555,657, BD Pharmingen). The gating strategy used and histograms of a representative donor are displayed inSupplementary Figure S1 . Flow cytometry was performed on the FACS Canto II (BD Biosciences) and analysed via FlowJo software (v10.8.2).
7
Flow Cytometric Phenotyping of Dendritic Cells
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For flow cytometry analysis, 2 x105 DCs were harvested by centrifugation, washed once in 2% FBS in PBS (Life Technologies) then resuspended in 100 µL 2% FBS in PBS. One microliter of FC blocker (BD Biosciences # 555404) was added to each sample and incubated for 15 min on ice. Sample viability was assessed using Fixable Viability Dye eFluor™ 780 (Thermo Fisher # 65–0865–14). Cells were then incubated with saturating concentrations of the different fluorochrome‐conjugated monoclonal antibodies (FITC‐conjugated anti‐CD86, APC‐conjugated CD80, PE‐conjugated anti‐CD11c, and PE‐Cy7‐conjugated anti‐MHCII (BD Biosciences) for 30 minutes at 4°C. The stained cells were washed twice in PBS and fixed in 1% paraformaldehyde PBS solution until analysis by flow cytometry. Flow cytometry was performed with the BD FACS Canto flow cytometer. Data were collected with FACS Diva software and analyzed using FlowJo V10. Cells were analyzed for geometric mean fluorescence intensity (mean fluorescence intensity of the antibody of interest/mean fluorescence intensity of isotype control) and for the percentage of marker‐positive cells. Over 10,000 cells per sample were analyzed.
8
Flow Cytometric Analysis of Dendritic Cell Maturation
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The iDC and mDC were washed with phosphate-buffered saline (PBS), aliquoted into fractions (5 × 105 cells/100 μl), and then stained for 30 min in the dark at room temperature with a final concentration of 5 μg/ml of the following antibodies purchased from BD Biosciences (San Jose, CA, USA): fluorescein isothiocyanate (FITC)-conjugated anti-CD11c (553801), FITC-conjugated anti-CD40 (553790), FITC-conjugated anti-CD80 (553768), and FITC-conjugated anti-CD86 (553691). As for the negative or no-antibody control, the cells were also stained with corresponding FITC-conjugated isotype-matched control antibodies or remained unstained. After staining, the cells were washed with PBS twice and analyzed by a BD LSRII flow cytometer (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star, San Carlos, CA, USA). The cells positive for FITC-CD11c were considered as DC that had successfully differentiated from bone marrow cells. The cells positive for FITC-CD40, FITC-CD80, and FITC-CD86 were considered as DC that had undergone successful maturation. For concurrent analysis of these molecules, the mDC were stained with phycoerythrin-indotricarbocyanine (PE-Cy7)-conjugated anti-CD11c (558079; BD Biosciences), together with the FITC-conjugated anti-CD40, FITC-conjugated anti-CD80, and FITC-conjugated anti-CD86, respectively.
9
Murine Liver Leukocyte Profiling
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RAW 264.7 cell lines were obtained from ATCC and cultured in RPMI-1640 containing 10% FBS. RAW 264.7 cells were treated with TNFα monoclonal antibody (MP6-XT3) and rat IgG1 kappa isotype control. Mouse leukocytes in the liver were purified as previously described. 18 The resulting leukocytes were incubated for 30 min at room temperature with BV421-conjugated anti-CD45 (BD), PE-Cy7 conjugated anti-CD11b (BD), APC-Cy7-conjugated live/dead, APC-conjugated anti-F4/80 (BD), FITC-conjugated anti-CD86 (BD), PE-conjugated anti-CD206 (BD) and PerCP-Cy5.5-conjugated anti-Ly6G (BD). Incubation was performed in the dark. The results were analyzed using a BD FACSCalibur flow cytometer and FlowJo software (Treestar, Ashland, OR, USA).
10
Flow Cytometry of Macrophage Markers
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The antibodies used in flow cytometry were APC-conjugated anti-F4/80, PE-cy7-conjugated anti-SAA, FITC-conjugated anti-CD86 and PE-conjugated anti-CD163 (Becton-Dickinson Biosciences). Data were analyzed using FlowJo software (Flowjo LLC, Ashland, OR, USA).
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