Single-cell suspensions from the SVF of fat tissue, spleen, and blood samples were incubated in FcBlock (BD Biosciences) for 15 min at 4°C. The cells were then stained with fluorophore-conjugated antibodies for 20 min at 4°C in the dark. The antibodies were purchased from BD Biosciences (CD8-FITC, CD11c-FITC, NK1.1-FITC, CD4-PE/Cy7, CD45.2-APC), eBioscience (San Diego, CA) (NKG2D-PE, F4/80-PE/Cy7, CD11b-APC/eFluor780), or BioLegend (San Diego, CA) (CD3ε-Pacific Blue). Dead cells were excluded with 7-AAD staining (BD Biosciences). Flow cytometry data was acquired with FACSCantoII or LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo (Treestar, Ashland, OR).
Cd11c fitc
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD11c-FITC is a fluorescently-labeled monoclonal antibody that binds to the CD11c surface antigen. It is commonly used in flow cytometry applications for the identification and analysis of dendritic cells and other CD11c-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd80 pe, by Thermo Fisher Scientific (6 mentions)
Cd4 fitc, by Thermo Fisher Scientific (5 mentions)
Cd86 pe, by Thermo Fisher Scientific (4 mentions)
Gr 1 pecy7, by Thermo Fisher Scientific (3 mentions)
Cd25 pe, by Thermo Fisher Scientific (3 mentions)
Cd86 apc, by Thermo Fisher Scientific (3 mentions)
Facscalibur, by BD (3 mentions)
Nk1.1 fitc, by Thermo Fisher Scientific (2 mentions)
Cd4 pe cy7, by Thermo Fisher Scientific (2 mentions)
F4 80 pe, by Thermo Fisher Scientific (2 mentions)
B220 percp cy5, by Thermo Fisher Scientific (2 mentions)
Cd4 v500, by BD (2 mentions)
Cd44 efluor450, by Thermo Fisher Scientific (2 mentions)
Cd8 efluor605, by Thermo Fisher Scientific (2 mentions)
Biotinylated antibodies against cd3, by Thermo Fisher Scientific (2 mentions)
B6 rag mice, by Jackson ImmunoResearch (2 mentions)
Anti cd16 32 2.4g2 and normal mouse sera, by Jackson ImmunoResearch (2 mentions)
Cd11b pe tr, by Thermo Fisher Scientific (2 mentions)
Cd3 alexa fluor 700, by Thermo Fisher Scientific (2 mentions)
Apc cd80, by Thermo Fisher Scientific (2 mentions)
29 protocols using cd11c fitc
1
Multiparameter Phenotyping of Immune Cells
2
Lung Inflammatory Cell Profiling by Flow Cytometry
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The composition of lung inflammatory cells was determined by flow cytometry (BD FacsCalibur Milan, Italy) using the following antibodies: CD11c-FITC, CD11c-APC, CD11b-PeCy5.5, Gr1-APC, CD3-PeCy5.5, CD4-FITC, CD25-PE, FoxP3-PeCy5.5, F4/80-PE, CD169-FITC, Arg I-PerCp (eBioscience, CA, USA). BAL-derived macrophages were stained for Tetramethyl rhodamine Esther (TMRE, 5 nM, Life Technologies, Monza, Italy) to measure mitochondrial membrane potential alterations. In another set of experiments BAL-derived macrophages were stained for MitoSOX Mitochondrial Superoxide Indicator as indicated in the manufacturer's guide (Life Technologies, USA).
3
Flow Cytometry Analysis of Mouse Tumor Tissue
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Small samples of mouse tumor tissues (2–3 wk after last immunization) were chopped in 1 ml of cell dissociation solution (1% 100 mg/ml collagenase/dispase, 0.5% 20 mg/ml DNase in DMEM) and incubated for 30 min at 37°C. The cell suspension (1×105 to 1×106 cells) was stained for use in flow cytometry after blocking the Fc receptors by applying ultra-blocking buffer (10% normal mouse serum, 10% normal hamster serum, 10% normal rat serum, and monoclonal Ab 2.4G2 in PBS). Abs used were CD11c-FITC (eBioscience, San Diego, CA, USA), CD45-PE-Texas-Red (Invitrogen, Carlsbad, CA, USA), F4/80-PE-Cy5 (eBioscience), CD11b-biotin (Miltenyi, Bergisch Gladbach, Germany), Gr-1-PE-Cy7 (eBioscience), and SA-PerCP-Cy5.5 (eBioscience). All samples were filtered through a 200 μm nylon mesh before running in BD FACSAria. The results were analyzed by using the Flowjo 10 program (Tree Star, Inc., Ashland, OR, USA).
4
Characterizing Microbiome-Host Interactions
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EcN and Salmonella typhimurium were purchased from China General Microbiological Culture Collection Center. Caco-2 and Raji cell lines were obtained from the American Type Culture Collection. Baker’s yeast was obtained from Lesaffre. Minimum Eagle’s medium (MEM), RPMI 1640, fetal bovine serum, and antibiotic/antimycotic solution were purchased from Thermo Fisher Scientific. Calcofluor-white stain was purchased from Sigma-Aldrich. The β-Glucan assay kit was purchased from Megazyme. Fluorescence-activated cell sorting antibodies including anti-mouse CD3-PerCP Cy5.5, CD4–fluorescein isothiocyanate (FITC), CD8–allophycocyanin (APC), CD11c-FITC, CD80–phycoerythrin (PE), CD86-APC, CD45R/B220-PE-Cy7, and CD138-APC were purchased from eBioscience. IgA-PE was purchased from Thermo Fisher Scientific. Plasmids pBBR1MCS2-Tac-mCherry (kanamycin resistance) and all other reagents were purchased from domestic suppliers and used as received.
5
Adoptive Transfer of Mpzl3-Deficient Lymphocytes
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Bone marrow was flushed out of femurs and tibias of Mpzl3 −/− or +/+ littermates (6 weeks old). The bone marrow was labeled using biotinylated antibodies against CD3, CD4, CD8 and B220 (eBioscience, San Diego, CA). Streptavidin-labeled microbeads were then used to magnetically deplete the biotinylated lymphocytes using autoMACS (Milteyni Biotec, Inc., Auburn, CA). 3 × 106 bone marrow cells after depletion were injected intraperitoneally into 2-day old B6 Rag −/− mice (The Jackson Laboratory, Bar Harbor, ME). After 10 weeks, the inguinal lymph nodes and spleens of these mice were analyzed by flow cytometry, and skin analyzed by histology. For flow cytometry, 3 × 106 cells were blocked using a cocktail of anti-CD16/32 (2.4G2) and normal mouse sera (Jackson ImmunoResearch, West Grove, PA) before being stained with fluorescently labeled antibodies to determine their phenotype and activation status. The following antibody conjugates were used: CD11b PE-TR (Life Technologies Corp.), CD4 V500 (Becton Dickinson, San Jose, CA), CD11c FITC, Gr-1 PECy7, CD3 Alexa Fluor 700, CD8 efluor605, B220 PerCpCy5.5, CD44 efluor450, CD62L APC, CD69 PE (eBioscience). Cells were then washed and re-suspended in 2% FBS in PBS for analysis with flow cytometers (LSR-II and Fortessa, Becton Dickinson).
6
Quantifying Tumor Immune Response
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To detect the in vivo immune response, 4T1 xenotransplant tumors were divided into eight groups and received treatments identical to in vivo therapeutic evaluation. On the 9th day, both primary tumors and distant metastases were harvested for single-cell suspensions fabrication. The prepared cells were further stained with CD11c-FITC (eBioscience, Catalog: N418), CD86-PE (eBioscience, Catalog: GL1), CD80-APC (eBioscience, Catalog: 16-10A1), F4/80-APC (Biolegend, Catalog: BM8), CD11b-PE (Biolegend, Catalog: M1/70), CD80-FITC (Biolegend, Catalog: 16-10A1), CD206-FITC (Biolegend, Catalog: C068C2), CD3-FITC (Biolegend, Catalog:17A2), CD8a-APC (Biolegend, Catalog: QA17A07) and CD4-PC5.5 (Biolegend, Catalog: RM4-5) antibody and then analyzed by flow cytometry. Serum was collected from different groups of mice, and cytokines including IL-6, IL-12, TNF-α and IL-10 were analyzed using ELISA kits according to the manufacturer's protocols. In addition, immunofluorescence staining was further conducted to investigate the infiltrating immune cells in tumor tissues.
7
Safflower Polysaccharide Inhibits Colitis-Associated Colorectal Cancer
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Dextran sulfate sodium (DSS) and azoxymethane (AOM) were obtained from Yeasen Biotechnology Co., Ltd. (Shanghai, China). Antibodies of p-IκBα, p-p65, IκBα, p65, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, United States). Human TNF-α and the IL-6 ELISA kit were obtained from eBioscience (Vienna, Austria). Anti–CD16-PE, CD11c-FITC, CD11b-FITC, F4/80-PE, CD-3-APC, CD80-PE, and CD206-PE were obtained from eBioscience (Minneapolis, MN, United States). The Annexin V-FITC/PI apoptosis detection kit was obtained from BD Biosciences (San Jose, CA, United States). Calcein-AM/PI was acquired from Beyotime (Haimen, China). PDTC was purchased from MedchemExpress (Monmouth Junction, NJ, United States). Safflower polysaccharide (SPS) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).
8
Characterization of Immune Cell Subsets in Murine Tumor Models
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On the 14th day after inoculation, mice were sacrificed and spleens and tumors were removed. Splenocytes were collected and stained with a combination of antibodies specific for CD11c-FITC, CD8α-PE, CD86-APC, CD80-PerCP-Cy5.5 (all purchased from eBioscience, San Diego, CA, USA). Splenocytes were then incubated in the dark at 4°C for 30 minutes and washed twice with FACS buffer (0.1% BSA and 0.05% sodium azide in PBS). Flow cytometry was performed using a BD FACS Arial flow cytometer and the results were analyzed with Flow-Jo software (Tree Star, Inc.).
9
Flow Cytometric Profiling of Lung Cells
10
Immune Cell Phenotyping Protocol
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The following antibodies and their corresponding isotype controls (all purchased from eBioscience, USA) were used for staining: CD4-Percp, Foxp3-FITC, CD11c-FITC, CD80-PE, MHCII-PE, PD-L1-PE, F4/80-FITC. CFSE were obtained from Invitrogen, USA. Cells were washed, fixed and stained according to the manufacturer's instructions. Samples were run on a FACSCalibur (BD Biosciences) and analyzed using FlowJo software (TreeStar).
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