Cells were stained with the following mAbs (BD Pharmingen, San Diego, CA, USA) and subjected to FACS analysis: FITC-conjugated anti-CD80 (Hamster, clone 16-10A1), and PE-conjugated anti-TLR4/MD2 (Rat, clone MTS510). Cells were acquired on a FACScan cytometer (BD Biosciences, San José, CA, USA) and data were analyzed by using CellQuest software (BD Immunocytometry Systems, San José, CA, USA).
Fitc conjugated anti cd80
Manufactured by BD
Sourced in United States
FITC-conjugated anti-CD80 is a laboratory reagent that binds to the CD80 surface antigen expressed on certain immune cells. The FITC (fluorescein isothiocyanate) fluorescent label allows for the detection and analysis of CD80-positive cells using flow cytometry or other compatible techniques.
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Lab products found in correlation
Fitc conjugated anti cd40, by BD (2 mentions)
Cellquest software, by BD (1 mentions)
Facscan cytometer, by BD (1 mentions)
Pe conjugated anti cd14, by BD (1 mentions)
Pe conjugated anti cd69, by BD (1 mentions)
Summit v4, by Agilent Technologies (1 mentions)
Pe conjugated anti cd86, by BD (1 mentions)
Pe conjugated anti hla dr, by BD (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Fluorescein isothiocyanate (fitc), by BD (1 mentions)
Fitc conjugated anti cd86, by BD (1 mentions)
Fitc conjugated anti cd11c, by BD (1 mentions)
Pe cy7 conjugated anti cd11c, by BD (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
4 protocols using fitc conjugated anti cd80
1
Flow Cytometric Analysis of CD80 and TLR4
2
Characterization of Monocyte Phenotype
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To study surface molecules expression, cells were washed with PBS, removed with a cell scraper, and labeled with human FITC-conjugated anti-CD14 (BioLegend, USA) or PE-conjugated anti-CD14 (BD Pharmingen, USA) and PE-conjugated anti-HLA-DR (BD Pharmingen, USA), PE-conjugated anti-CD86 (BD Pharmingen, USA) or FITC-conjugated anti-CD80 (BD Pharmingen, USA), and FITC-conjugated anti-CD40 (BD Pharmingen, USA) or PE-conjugated anti-CD69 (BD Pharmingen, USA) for 30 minutes at 4°C.
Cells were then washed twice with PBS + FCS 5% (200 ×g, 7 minutes) and resuspended in the same solution in the cold. Fluorescence measurements were performed using a FACScan or a FACSCalibur flow cytometer (Beckton, Dickinson and Company, USA). Fluorescence signals for no less than 10,000 cells were recorded in the monocyte gate, which was determined according to cell size and complexity parameters. Using these parameters, approximately 98% of the cells in this gate were CD14+. Data analyses were performed using Summit v4.3 software (Dako, USA).
Cells were then washed twice with PBS + FCS 5% (200 ×g, 7 minutes) and resuspended in the same solution in the cold. Fluorescence measurements were performed using a FACScan or a FACSCalibur flow cytometer (Beckton, Dickinson and Company, USA). Fluorescence signals for no less than 10,000 cells were recorded in the monocyte gate, which was determined according to cell size and complexity parameters. Using these parameters, approximately 98% of the cells in this gate were CD14+. Data analyses were performed using Summit v4.3 software (Dako, USA).
3
Phenotypic Analysis of Stimulated moDCs
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MoDCs stimulated with six Introd-Vac-strains or six ACV-strains were used to analyze surface marker expression. These moDC were stained with PE-conjugated anti-CD101 (BD Biosciences), APC-conjugated anti-CD97 (R&D systems), FITC-conjugated anti-CD80 (BD Biosciences), Pacific Blue-conjugated anti-CD86 (BioLegend), PerCP-eFluor710- conjugated anti-CD274, PE/Cy7-conjugated anti-CD172a/b and with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) for 30 minutes at 4°C. The moDC were washed twice with FACS buffer (PBS pH 7.2; 0,5% BSA; 0,5 mM EDTA) and fixed with 1.5% PFA. Data was acquired on FACS Canto II (BD Biosciences) and analyzed using FlowJo software (Tree Star). More than 90% of the stimulated cells remained viable after the 48-hour stimulation.
4
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.
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