Isolated DCs were cultured for 24 h in the presence of Rv0315 or mutant protein (5, 10 or 20 μg/ml). These BMDCs were harvested, washed with phosphate-buffered saline (PBS) and stained with APC-conjugated anti-H-2Kb (MHC class I), FITC-conjugated anti-I-A/I-E (MHC class II), PE-conjugated anti-CD80, and APC-conjugated anti-CD86, along with PE-Cy7-conjugated anti-CD11c (BD Pharmingen) for 30 min at 4 °C. The cells were then washed three times with PBS and resuspended in 500 μl of PBS. Surface markers for BMDC maturation were analyzed by FACSCalibur flow cytometry (BD Biosciences). DCs were stimulated with LPS in medium as a positive control or with medium alone as an untreated control. Cytotoxicity was analyzed by staining for surface-exposed phosphatidylserine with FITC-annexin V in combination with propidium iodine (PI) (BD Biosciences) according to the manufacturer’s instructions.
Pe cy7 conjugated anti cd11c
Manufactured by BD
Sourced in United States
PE-Cy7-conjugated anti-CD11c is a fluorescently-labeled monoclonal antibody that binds to the CD11c protein, which is expressed on the surface of dendritic cells and certain other immune cells. This product can be used for the identification and analysis of cell populations expressing CD11c in flow cytometry applications.
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Lab products found in correlation
Propidium iodine, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Novocyte flow cytometer, by Agilent Technologies (1 mentions)
Fitc conjugated anti cd11b, by BD (1 mentions)
Apc conjugated anti f4 80, by BioLegend (1 mentions)
Pe conjugated anti il 4, by BD (1 mentions)
Fitc conjugated anti ifn γ, by BD (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Pacific blue conjugated anti cd4, by BioLegend (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Cd16 32, by BD (1 mentions)
Pe conjugated anti cd4, by BD (1 mentions)
Bv605 conjugated anti cd86, by BD (1 mentions)
Pe conjugated anti cd40, by BD (1 mentions)
Fitc conjugated anti cd8a, by BD (1 mentions)
Apc cy7 conjugated anti cd3, by BD (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
7 aminoactinomycin d, by BD (1 mentions)
Apc conjugated anti cd4, by BD (1 mentions)
6 protocols using pe cy7 conjugated anti cd11c
1
Rv0315 Protein Modulates Dendritic Cell Maturation
2
Isolation and Characterization of Immune Cells from Mesenteric Lymph Nodes
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Cell suspensions were prepared from mesenteric lymph nodes of KKAy mice according to previous report (Moro et al., 2015 (link)). Single-cell suspensions were stained with antibodies against the following cell surface antigens: fluorescein isothiocyanate (FITC)-conjugated anti-CD11b (557396; BD, USA), phycoerythrin (PE)-conjugated anti-MHC-II (107608; Biolegend, USA), PE-CY7-conjugated anti-CD11c (558097; BD, USA), PE-CY5.5-conjugated anti-CD45.2 (109828; Biolegend, USA), and allophycocyanin (APC)-conjugated anti-F4/80 (123116; Biolegend, USA). Samples were detected on a NovoCyte flow cytometer (ACEA Biosciences, China), and the data were analyzed with NovoExpress flow cytometry analysis software (ACEA Biosciences, China).
3
Intracellular IL-12 Expression in BMDC
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The expression of intracellular IL-12 was analyzed in BMDC after 24 hours of stimulation with LPS (1 µg/ml) at 37°C, 5% CO2, in the presence or absence of 10 µM CA074Me or 10 µM ZRLR, and brefeldin A (3 µg/ml, eBioscience). The cells were then incubated for 20 min in 4% PFA fixation buffer, permeabilized for 20 min at 4°C using 0.1% saponin, 1% FCS permeabilization buffer, and incubated for 1 hour with (PECy7)-conjugated anti-CD11c and PE-conjugated anti-IL-12(p40/p70, BD Biosciences. Data was obtained using the MACSQuant flow cytometer. Furthermore, cells from polarization assays described below were fixed with 2% formaldehyde for 20 min at 4°C, permeabilized for 20 min at 4°C, and stained with the following Ab diluted in permeabilization buffer: Pacific Blue-conjugated anti-CD4 (Biolegend), FITC-conjugated anti-IFN-γ (BD Biosciences), PE-conjugated anti-IL-4 (BD Biosciences), and allophycocyani-conjugated anti-IL10 (Biolegend). Data was obtained using a LSR-II flow cytometer (BD Biosciences, San Jose, USA). All results were analyzed using the software FlowJo.
4
Multi-Color Flow Cytometry Analysis
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Six- and four-color flow cytometry were performed on BD LSRFortessa X-20. We adjusted voltages based on unstained cells, and compensation was performed according to single-stained-positive control for each color. Dead cells were excluded by 7-aminoactinomycin D (BD Biosciences) and added 0.5 µl CD16/32 (BD Biosciences) to block nonspecific Fc-mediated interactions.
Six-color staining was performed by using the following panel of mAbs to quantitate DCs immunophenotyped: PE-Cy7 conjugated anti-CD11c (BD Biosciences, San Diego, CA, USA), BV421 conjugated anti-CD80 (BD Biosciences, San Diego, CA, USA), BV605 conjugated anti-CD86 (BD Biosciences, San Diego, CA, USA), PE conjugated anti-CD40(BD Biosciences, San Diego, CA, USA), Alexa Flour488 anti-MHCII(BD Biosciences, San Diego, CA, USA).
Four-color staining was performed to address the proliferation levels of T lymphocytes by using the following panel of mAbs: APC-Cy7 conjugated anti-CD3 (BD Biosciences, San Diego, CA, USA), PE conjugated anti-CD4 (BD Biosciences, San Diego, CA, USA), FITC conjugated anti-CD8a (BD Biosciences, San Diego, CA).
Six-color staining was performed by using the following panel of mAbs to quantitate DCs immunophenotyped: PE-Cy7 conjugated anti-CD11c (BD Biosciences, San Diego, CA, USA), BV421 conjugated anti-CD80 (BD Biosciences, San Diego, CA, USA), BV605 conjugated anti-CD86 (BD Biosciences, San Diego, CA, USA), PE conjugated anti-CD40(BD Biosciences, San Diego, CA, USA), Alexa Flour488 anti-MHCII(BD Biosciences, San Diego, CA, USA).
Four-color staining was performed to address the proliferation levels of T lymphocytes by using the following panel of mAbs: APC-Cy7 conjugated anti-CD3 (BD Biosciences, San Diego, CA, USA), PE conjugated anti-CD4 (BD Biosciences, San Diego, CA, USA), FITC conjugated anti-CD8a (BD Biosciences, San Diego, CA).
5
Analysis of Myeloid Cells and T Cells in BALF
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BALF samples were collected by making an incision in the trachea and lavaging the lung twice with 0.8 ml PBS, pH 7.4. Total leukocyte counts were determined using a hemacytometer.
For flow cytometric analysis, BALF cells were incubated with 2.4G2 mAb against FcγRII/III, and stained with PE-Cy7-conjugated anti-CD11c (BD Biosciences), APC-Cy7-conjugated anti-CD11b (BD Biosciences), PE-conjugated Ly6B (Abcom), PCP-Cy5-conjugated anti-Ly6C (eBiosciences) and PE-Cy7 conjugated anti-Ly6G mAb (BD Biosciences) for myeloid cell analysis. APC-conjugated anti-CD11c, APC-Cy7-conjugated anti-CD11b (BD Biosciences), PE-conjugated anti-MHC II (BD Biosciences) and PCP-Cy5-conjugated anti-F4/80 (eBiosciences) were used for DC analysis. PCP-Cy5-conjugated anti-CD3 (Biolegend), APC-conjugated anti-CD4 (BD Biosciences) and APC-Cy7-conjugated anti-CD8 mAb (BD Biosciences) were used for T cell analysis. Splenocytes and BALF cells were stained with Tetramer Alexa 647-Labeled H-2D(b)/PA224(SSLENFRAYV) and BV421-Labeled H-2D(b)/NP366 (ASNENMETM), using FITC-conjugated anti-CD3 (BD Biosciences), APC-Cy7-conjugated anti-CD4 (BD Biosciences) and PE-conjugated anti-CD8 mAb (BD Biosciences) for cell surface markers. The stained cells were analyzed on a BD FACSCanto or BD LSRII-green using FlowJo and BD FACSDiva software analysis.
For flow cytometric analysis, BALF cells were incubated with 2.4G2 mAb against FcγRII/III, and stained with PE-Cy7-conjugated anti-CD11c (BD Biosciences), APC-Cy7-conjugated anti-CD11b (BD Biosciences), PE-conjugated Ly6B (Abcom), PCP-Cy5-conjugated anti-Ly6C (eBiosciences) and PE-Cy7 conjugated anti-Ly6G mAb (BD Biosciences) for myeloid cell analysis. APC-conjugated anti-CD11c, APC-Cy7-conjugated anti-CD11b (BD Biosciences), PE-conjugated anti-MHC II (BD Biosciences) and PCP-Cy5-conjugated anti-F4/80 (eBiosciences) were used for DC analysis. PCP-Cy5-conjugated anti-CD3 (Biolegend), APC-conjugated anti-CD4 (BD Biosciences) and APC-Cy7-conjugated anti-CD8 mAb (BD Biosciences) were used for T cell analysis. Splenocytes and BALF cells were stained with Tetramer Alexa 647-Labeled H-2D(b)/PA224(SSLENFRAYV) and BV421-Labeled H-2D(b)/NP366 (ASNENMETM), using FITC-conjugated anti-CD3 (BD Biosciences), APC-Cy7-conjugated anti-CD4 (BD Biosciences) and PE-conjugated anti-CD8 mAb (BD Biosciences) for cell surface markers. The stained cells were analyzed on a BD FACSCanto or BD LSRII-green using FlowJo and BD FACSDiva software analysis.
6
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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