Dopamine, SCH58261, Collagenase A, 2′,7′‐Dichlorofluorescein diacetate (H2DCF), and Coformycin (Erythro‑9‑(2‑hydroxy‑3‑nonyl) adenine) were purchased from Sigma‐Aldrich. 4‐bromomethyl phenylboronic acid (BPBA), Amino polyethylene glycol (PEG), Rhodamine B isothiocyanate (Rhodamine B), and Fluorescein5(6)‐isothiocyanate (FITC) were acquired from Aladdin Industrial Corporation. Bodipy and Live/Dead staining kits were obtained from Thermo Fisher Scientific. Adenosine, CCK‐8, FITC‐Annexin V Apoptosis Detection Kit, Enhanced ATP Assay Kit, BCA assay kit, and Protease inhibitor cocktail for general use (100X) were purchased from Beyotime Biotechnology. Percp‐CRT antibody was purchased from StressMarq Biosciences Inc. Antibodies of APC‐CD73, Alexa Fluor 488‐HMGB1, CD16/32, Foxp3, APC‐CD11c, FITC‐CD80, PE‐CD86, PE‐CD40, FITC‐MHCII, FITC‐CCR7, PE‐CD45, PEcy7‐Gr‐1, APC‐CD11b, APC‐CD3, FITC‐CD4, PEcy7‐CD8α, and PE‐IFNγ were purchased from BioLegend. The antibody of A2AR was obtained from Proteintech Group, Inc. Adenosine ELISA Kit was obtained from Shanghai Jianglai Biological Technology Co., Ltd. ELISA kits of TNF‐α, IL‐6, IFN‐γ, and IL‐12p40 were purchased from Dakewe Biotech Co., Ltd. Cytokines of GM‐CSF, IL‐4, and IL‐2 were purchased from Shanghai Jinan Technology Co., Ltd. DNase I and Bouin's solution were purchased from Beijing solarbio science&technology co., ltd.
Cd11c apc
Manufactured by BioLegend
Sourced in United States
CD11c-APC is a fluorescent-labeled antibody used to detect and analyze the expression of the CD11c cell surface marker. CD11c is a type I transmembrane glycoprotein that serves as a component of the integrin αx subunit, which is primarily expressed on the surface of dendritic cells, macrophages, and granulocytes. The APC (Allophycocyanin) fluorescent label allows for the detection and quantification of CD11c-positive cells using flow cytometry or other fluorescence-based analysis techniques.
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Cd3 fitc, by BioLegend (4 mentions)
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Cd11b apc, by BioLegend (4 mentions)
71 protocols using cd11c apc
1
Immunomodulation Pathway Evaluation
2
Comprehensive Tumor Immune Profiling in Mice
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CT26 tumor-bearing BALB/c mice were randomly assigned into separate groups (n = 4 per group) and i.v. administrated with NP/mDs and each component at a Pt dose of 2.5 mg/kg, and/or i.p. injected with αPD-L1 at a dose of 100 μg/mouse. After a Q3D × 5 schedule, all mice were euthanized in accordance with institutional policy, and tumors were harvested, cut into pieces, treated with collagenase IV, and ground with the rubber end of syringes.
Singlet suspensions were incubated with CD16/CD32 antibody, followed by incubation with PerCP Cy5.5-CD45, PE Cy7-CD3ϵ, FITC-CD4, PE-CD8a, APC-CD25, PE-Foxp3, APC-CD11c, FITC-CD80, PE-CD86, FITC-F4/80, and APC-CD206 antibodies (Biolegend, USA), respectively. Cell populations were sorted using flow cytometry and data were analyzed using FlowJo X.
Singlet suspensions were incubated with CD16/CD32 antibody, followed by incubation with PerCP Cy5.5-CD45, PE Cy7-CD3ϵ, FITC-CD4, PE-CD8a, APC-CD25, PE-Foxp3, APC-CD11c, FITC-CD80, PE-CD86, FITC-F4/80, and APC-CD206 antibodies (Biolegend, USA), respectively. Cell populations were sorted using flow cytometry and data were analyzed using FlowJo X.
3
Comprehensive Immune Cell Profiling
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PBMCs from above stimulation were collected and washed with PBS for twice. The cells were then treated with blocker and incubated on ice for 15 min. Samples were stained with antibodies and incubated in the dark at 4°C for 20 min. Afterwards, cells were washed with PBS and then centrifuged again. After centrifugation, the cells were re-suspended with PBS and ready for FACS analysis. The antibodies were used as follows: APC-750-CD45, PC7-CD11b, PE-CY5-HLA-DR, APC-CD11C, FITC-CD206, PE-CD80, PC7-CD19, BV510-CD56, APC-CD3, FITC-CD4, PE-CD8 (BioLegend, San Diego, CA.).
4
Multicolor Flow Cytometry Panel
5
Multiparametric Flow Cytometry of Immune Cells
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For cell surface staining, cells were incubated with anti-mouse FITC-CD11b (Biolegend), APC-CD11c (Biolegend), PE-CD80 (Biolegend), PE-CD86 (Biolegend), and PE-MHCII (Biolegend) after FcR blocking. Isotype-matched mouse immunoglobulin served as control. For intracellular cytokine staining, splenocytes were plated into a 12-well culture plate at a density of 4 × 106/mL and stimulated with 10 μg/mL CAIX protein at 37°C and 5% CO2 for 72 h. These cells were also stimulated with 500 ng/mL Ionomycin (Sigma-Aldrich) and 50 ng/mL PMA (Sigma-Aldrich) plus 5 ng/mL Brefeldin A (BFA, eBioscience) for the last 5 h. Cell surface staining was performed on the stimulated splenocytes or isolated TILs with anti-mouse PerCP-Cy5.5-CD8α (BD Pharmingen), and then intracellular staining was performed with anti-mouse APC-IFN-γ (BD Pharmingen), PE-IL-2 (BD Pharmingen), and FITC-TNF-α (BD Pharmingen). FlowJo software (Tree Star Inc) analyzed the sample data acquired from BD FACSCanto II (BD Biosciences) in FACSDiva software (BD Biosciences).
6
Murine Dendritic Cell and Macrophage Characterization
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Six-week-old female C57BL/6 mice were purchased from Beijing Vital River Lab Animal Technology Co., Ltd. Dendritic cells (DCs) were isolated according to previously published methods (22 (link)). Macrophages were obtained from mouse bone marrow-derived cells. The cells were cultured in 1640 medium supplemented with 10 ng/mL M-CSF and 10% FBS in 6-well plates for 6 days with the medium changed every 2 days.
Isolated dendritic cells or macrophages were exposed to VLPs (10μg) or mock treated for 48 hours before assessing changes in the expression of cell surface molecules and their production of cytokines using flow cytometry and ELISA, respectively. The cells were stained with APC-CD11c, APC-F4/80, PE-CD80, FITC-CD86, FITC-MHC-II, and FITC-CD40 antibodies (all from Biolegend, San Diego, CA, USA) and changes in the percentage (%) of cells expressing these markers determined. The levels of cytokines (TNF-α, IL-6 and IL-12p70) were detected using cytokines ELISA kits purchased from R&D Systems (Minneapolis, MN, USA).
Isolated dendritic cells or macrophages were exposed to VLPs (10μg) or mock treated for 48 hours before assessing changes in the expression of cell surface molecules and their production of cytokines using flow cytometry and ELISA, respectively. The cells were stained with APC-CD11c, APC-F4/80, PE-CD80, FITC-CD86, FITC-MHC-II, and FITC-CD40 antibodies (all from Biolegend, San Diego, CA, USA) and changes in the percentage (%) of cells expressing these markers determined. The levels of cytokines (TNF-α, IL-6 and IL-12p70) were detected using cytokines ELISA kits purchased from R&D Systems (Minneapolis, MN, USA).
7
Phenotypic Characterization of MDSCs in Liver Cancer
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MDSCs from the spleens of orthotopic H22 liver cancer-bearing mice were isolated by MACS technique (Miltenyi Biotec). Approximately 1 × 10^6 MDSCs were cultured in 24-well culture-plate and stimulated by JHD (250μg/ml, 500μg/ml) and oxaliplatin(1μg/ml, Solarbio) (Kim and Kim, 2019 (link)), respectively. After stimulating for 36 or 48 hours, cells were collected and their Fc-receptors were blocked with Fc-receptor blocking reagent (Miltenyi Biotec). The cells were then stained with APC-CD11c, PE-F4/80, FITC-CD80, and Percp/Cy5.5-MHCII antibodies (Biolegend) following the manufacturer's instructions and detected with Flow cytometry (Beckman Coulter Cytoflex). Cells not bound to fluorescent antibodies and cells bound to one fluorescent antibody were used for the fluorescent compensation which was performed by CytExpert software.
8
Generating Collagen-Specific T Cell Tetramers
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PE-conjugated MOG tetramer as well as biotinylated DR1 monomers were ordered from NIH Tetramer Core Facility (Atlanta, GA). PE-conjugated DR1/Collagen II (280–294) tetramers were created in-house using the CLIP/DR1 monomers and a NIH Tetramer Core Facility protocol. Briefly, the process involved cleavage of the CLIP peptide from the DR1 monomer and an exchange reaction with the collagen II (289–294) peptide. For the flow cytometry experiment, Brilliant Violet 421-CD44, Alexa Fluor 700-CD4, PE-Cy7-CD8, FITC-CD3, BV421-CD25, Alexa647-Foxp3, APC-B220, APC-CD11c, PE-Cy7-CD69, and APC-F4/80 from Biolegend (San Diego, CA) and fixable viability dye eFluor 780 from eBioscience (San Diego, CA) were used.
9
Isolation of Immune Cells from Murine Colon
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Colon collected from control or DSS-treated mice was cut into small pieces then digested with 2.5 mg/mL Collagenase D and 30 ng/mL DNAse1 for 40 min at 37 °C then filtered through a 70 µm filter to generate single cell suspension for FACS analyses. Cells were then stained with Fixable Viability Dye eFluor 506 (eBioscience, 65-0866-14), blocked with anti-mouse CD16/32 (eBioscience, 14016185), followed by staining with antibody cocktails for preadipocytes or immune cells. The antibody cocktail for immune cells includes FITC -CD45 (BioLegend, 103107), PECy7-CD11b (BioLegend, 101216), FITC-Ly6G (eBioscience, 11593182), PE-F4/80 (eBioscience,12480182), APC-CD11C (BioLegend, 117310), AF700-MHCII (eBioscience, 56532182), and APC-eFluro-CD4 (eBioscience, 47-0042-80) PE -CD19 (BioLegend, 115507). All antibodies were used at a final dilution of 1 to 100. FACS analyses for surface expression of immune cell markers were performed by the BD FACSCanto RUO machine and analyzed by FlowJo V10 software.
10
Multiparameter Flow Cytometry Panel
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A single-cell suspension was blocked with mouse FcR blocking reagent (Miltenyi Biotec, USA) at 4°C for 10 min prior to surface staining. For intracellular IFN-γ staining, the cells were fixed and permeabilized with Fixation/Permeabilization Kit (BD Biosciences, USA) and then stained with IFN-γ antibody. The following anti-mouse antibodies were used: PE-CD44 (clone:IM7), APC-CD24 (clone:M1-69), FITC-CD3e (clone: 145-2C11), PE-CD45 (clone:30-F11), APC-MHC class II (I-A/I-E) (clone: AMS-32.1), PE-CD86 (clone: GL 1) from eBioscience (San Diego, CA, USA); PE/Cy7-CD8a (clone: 53-6.7), PE-MHC class I (H-2Kd) (clone: AMS-32.1), PE/Cy7-CD40 (clone: 3-23), PE/Cy7-CD11c (clone: HL3) from BD Biosciences (USA); and APC-CD8a (clone: 53-6.7), PE-IFN-γ (clone: XMG 1.2), APC-CD11c (clone:N418), APC-CD80 (clone: 16-10A1) from Biolegend (USA). For ALDH1 staining, the ALDH1 activity was detected by the ALDEFLUOR kit (Stem Cell Technologies, Canada) according to the instructions of the manufacturer. Data were acquired on BD FACS Calibur (USA) and analyzed by the Flow-Jo software (Tree Star, OR) version 7.1.2. Fixable viability dye eFluor 520 (eBioscience, USA) was used to exclude dead cells.
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