Lung single-cell suspensions and BMDC suspensions collected from different groups were stained with PE anti-mouse CD11c (eBioscience, San Diego, CA, USA) and APC-Cy7 anti-mouse F4/80 (Biolegend Inc., San Diego, CA, USA). DCs were marked as CD11c+ and F4/80− cells. This was followed by analysis of the phenotype and the maturation of DCs using FITC anti-mouse CD80, FITC anti-mouse major histocompatibility complex II (MHCII), FITC anti-mouse CD40 (eBioscience, San Diego, CA, USA), and APC anti-mouse CD86 (Biolegend Inc., San Diego, CA, USA) stains. Among them, cDCs were marked as CD11c+MHCII+ double-positive cells. In addition, the cDCs2 (type 2 cDCs) in lung and spleen MNCs were stained as CD11c+CD11b+ double-positive cells using PE anti-mouse CD11c and APC anti-mouse CD11b (Biolegend Inc., San Diego, CA, USA). All the cells, after being washed with PBS, were analyzed using Flow Cytometer (FACSAria™ III, BD Biosciences, USA). The FlowJo software (FlowJo LLC, Ashland, Ore) was used to analyze the data.
Anti mouse cd11c pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-mouse CD11c-PE is a fluorescently-labeled antibody that binds to the CD11c cell surface antigen expressed on dendritic cells in mice. It is used for the identification and enumeration of CD11c-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 3, by BD (5 mentions)
Fitc anti mouse cd80, by Thermo Fisher Scientific (4 mentions)
Apc anti mouse cd11b, by BioLegend (3 mentions)
Apc cy7 anti mouse f4 80, by BioLegend (2 mentions)
Pe anti mouse cd11c, by BioLegend (2 mentions)
Apc anti mouse cd86, by BioLegend (2 mentions)
Fitc anti mouse major histocompatibility complex 2, by Thermo Fisher Scientific (2 mentions)
Fitc anti mouse cd40, by Thermo Fisher Scientific (2 mentions)
Software, by FlowJo (2 mentions)
Anti mouse mhc 2 fitc, by Thermo Fisher Scientific (2 mentions)
Anti mouse f4 80 apc, by Thermo Fisher Scientific (2 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (2 mentions)
Ebioscience fixable viability dye efluor 506, by Thermo Fisher Scientific (2 mentions)
Facsdiva software 6, by BD (2 mentions)
Fc receptor blocker, by Miltenyi Biotec (2 mentions)
Anti mouse cd11b pe cy7 clone m1 70, by Thermo Fisher Scientific (2 mentions)
Anti mouse cd24 fluorescein 5 isothiocyanate fitc clone m1 69, by Thermo Fisher Scientific (2 mentions)
Fcr blocking reagent, by Miltenyi Biotec (2 mentions)
Apc anti mouse mhc 2, by Thermo Fisher Scientific (1 mentions)
Xenogen ivis spectrum imaging system, by PerkinElmer (1 mentions)
12 protocols using anti mouse cd11c pe
1
Identifying Dendritic Cell Subsets
2
In Vivo Targeting and Immune Response of mRNA-loaded LNPs
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For the in vivo targeting study, male C57BL/6 mice (20 ± 2 g) were subcutaneously injected with Fluc mRNA-loaded 1.5PD-LNPs, PC-LNPs, and SAPC-LNPs (10 μg/mouse, n = 3). D-fluorescein potassium (3 mg/100 μL) was injected intraperitoneally at 24 h. The popliteal lymph nodes of the mice were collected for bioluminescence imaging 10 min later using a Xenogen IVIS Spectrum Imaging System (Perkin Elmer, USA).
Another group of male C57BL/6 mice weighing 20 ± 2 g were subcutaneously injected with EGFP mRNA loaded into 1.5PD-LNPs, PC-LNPs, SAPC-LNPs, or PBS at a dose of 10 μg/mouse. The popliteal lymph nodes of the mice were collected to prepare single cell suspensions. The lymph nodes were gently ground on 70 μm cell mesh strainers and then centrifuged at 4 °C for 10 min at a speed of 500 g. The cells were precipitated and resuspended in PBS. For each test, APC anti-mouse MHC-II (eBioscience) and PE anti-mouse CD11c (eBioscience) antibodies were added according to the protocol and incubated for 30 min at 4 °C. The cell precipitate was resuspended in PBS for flow cytometry analysis. The sections were stained with primary antibodies against CD169, followed by immunostaining using PE-conjugated secondary antibodies and DAPI staining to visualize the nuclei.
Another group of male C57BL/6 mice weighing 20 ± 2 g were subcutaneously injected with EGFP mRNA loaded into 1.5PD-LNPs, PC-LNPs, SAPC-LNPs, or PBS at a dose of 10 μg/mouse. The popliteal lymph nodes of the mice were collected to prepare single cell suspensions. The lymph nodes were gently ground on 70 μm cell mesh strainers and then centrifuged at 4 °C for 10 min at a speed of 500 g. The cells were precipitated and resuspended in PBS. For each test, APC anti-mouse MHC-II (eBioscience) and PE anti-mouse CD11c (eBioscience) antibodies were added according to the protocol and incubated for 30 min at 4 °C. The cell precipitate was resuspended in PBS for flow cytometry analysis. The sections were stained with primary antibodies against CD169, followed by immunostaining using PE-conjugated secondary antibodies and DAPI staining to visualize the nuclei.
3
Murine Dendritic Cell Phenotyping
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Following purification, MLNDCs were cultured in RPMI-1640 with 10% fetal bovine serum, 100 IU/mL penicillin and 100 ug/mL streptomycin and incubated at 37°C, 5% CO2.
Reagents: phosphate buffered saline (PBS; GNM-20012, Hangzhou, China), RPMI1640 and 10% fetal bovine serum (Gibco, Grand Island, NY, USA), PE-anti-mouse CD11c, APC-anti-mouse MHC-I, FITC-anti-mouse MHC-II, FITC-anti-mouse CD80, APC-anti-mouse CD86, PE-mouse IgG1 control, APC-mouse IgG2a control, FITC-mouse IgG2b control (eBioscience, Sandiego, California, USA), 4% paraformaldehyde (Sigma, St. Louis, Missouri, USA), blocking solution (Sigma), Anti-CRHR1 (Anbo Biotechnology, San Francisco, California, USA), Anti-CRHR2 (Anbo Biotechnology), FITC anti-Mouse CD11c (eBioscience), Cy3 conjugated goat anti-rabbit IgG (H+L), 4′,6-diamidino-2-phenylindole (DAPI) (Southern Biotech Associates, Birmingham, Alabama, USA), anti-fading agent (Sigma). Ten percent of SDS-PAGE (Amersham Biosciences, Piscataway, New Jersey, USA), polyvinylidene fluoride (PVDF) membranes (Amersham Biosciences), goat anti-rabbit MHC-II (Abcam, Cambridge, UK), HRP-conjugated secondary antibody(Abcam). Carboxyfluorescein diacetate succinimidyl ester (Invitrogen Ltd, Paisley, UK), anti-mouse CD4 antibody, and anti-mouse CD8 antibody (Invitrogen Ltd).
Reagents: phosphate buffered saline (PBS; GNM-20012, Hangzhou, China), RPMI1640 and 10% fetal bovine serum (Gibco, Grand Island, NY, USA), PE-anti-mouse CD11c, APC-anti-mouse MHC-I, FITC-anti-mouse MHC-II, FITC-anti-mouse CD80, APC-anti-mouse CD86, PE-mouse IgG1 control, APC-mouse IgG2a control, FITC-mouse IgG2b control (eBioscience, Sandiego, California, USA), 4% paraformaldehyde (Sigma, St. Louis, Missouri, USA), blocking solution (Sigma), Anti-CRHR1 (Anbo Biotechnology, San Francisco, California, USA), Anti-CRHR2 (Anbo Biotechnology), FITC anti-Mouse CD11c (eBioscience), Cy3 conjugated goat anti-rabbit IgG (H+L), 4′,6-diamidino-2-phenylindole (DAPI) (Southern Biotech Associates, Birmingham, Alabama, USA), anti-fading agent (Sigma). Ten percent of SDS-PAGE (Amersham Biosciences, Piscataway, New Jersey, USA), polyvinylidene fluoride (PVDF) membranes (Amersham Biosciences), goat anti-rabbit MHC-II (Abcam, Cambridge, UK), HRP-conjugated secondary antibody(Abcam). Carboxyfluorescein diacetate succinimidyl ester (Invitrogen Ltd, Paisley, UK), anti-mouse CD4 antibody, and anti-mouse CD8 antibody (Invitrogen Ltd).
4
Phenotypic Analysis of Dendritic Cells
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Lung single cell suspensions and BMDCs suspensions collected from different groups were stained with PE anti-mouse CD11c (eBioscience, San Diego, CA, USA) and APC-Cy7 anti-mouse F4/80 (Biolegend Inc., San Diego, CA, USA). DCs were marked as CD11c + and F4/80 -cells. This was followed by analysis of the phenotype and the maturation of DCs using FITC anti-mouse CD80, FITC anti-mouse major histocompatibility complex II (MHCII), FITC anti-mouse CD40 (eBioscience, San Diego, CA, USA) and APC anti-mouse CD86 (Biolegend Inc., San Diego, CA, USA) stains. Among them, cDCs were marked as CD11c + MHCII + double positive cells. In addition, the cDCs2 (type 2 cDCs) in lung and spleen MNCs were stained as CD11c + CD11b + double positive cells using PE anti-mouse CD11c and APC anti-mouse CD11b (Biolegend Inc., San Diego, CA, USA). All the cells, after being washed with PBS, were analyzed using Flow Cytometer (FACSAria™ III, BD Biosciences, USA). The FlowJo software (FlowJo LLC, Ashland, Ore) was used to analyze the data.
5
Multiparametric Flow Cytometry of Murine Liver Cells
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Single cell suspensions of livers were washed in PBS prior to viability staining (Zombie Near-InfraRed) and staining for surface proteins for 30 minutes. Excess antibody was washed out with PBS prior to sample fixation. All antibodies were used at a 1:200 dilution, except F4/80-PE-Cy7 which was used at 1:150, for 30 minutes on ice. Samples were analyzed using a BD LSR-II (4 lasers) and a Cytek Aurora (4 lasers). Antibodies used for flow cytometry included the following: BV421 anti-mouse F4/80 (BD, 100433), eFluor 506 anti-mouse CD45 (Invitrogen, 69-0451-82), FITC anti-mouse Ly6G (BD, 553127), PerCP/Cyanine5.5 anti-mouse CD4 (Biolegend, 12-0114-81), PE anti-mouse CD11c (Invitrogen, 25-0032-82), PE Cyanine 7 anti-mouse CD3 (Invitrogen, 565411), APC anti-mouse CD11b (Biolegend, 101211) and live/dead near-IR dead cell stain kit (Invitrogen, L34975).
6
Tumor Immune Cell Profiling
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Tumors were collected at 15 days after s.c. injection of B16F10 cells. The tumor masses were minced into 3–5 mm2 slices and dissociated by incubating in 1 mg/ml collagenase type IV (Sigma, C5138) and 20 mg/ml DNase (Sigma, D5025) for 30 min with rotation. The resulting mixture was suspended in FACS buffer (5% FBS in PBS) and passed through a 70-μm nylon cell strainer (SPL, 93070) to obtain a single-cell suspension. Fc receptors were blocked using TruStain FcX (Biolegend, 101319) according to the manufacturer’s protocol, and cells were stained with the following fluor-conjugated antibodies for 1 h on ice: PE anti-mouse CD8 (Invitrogen, 12-0081-82), PerCP anti-mouse CD3 (Invitrogen, 45-0031-82), PE anti-mouse NK1.1 (Invitrogen, 12-5941-82), PE anti-mouse CD11c (Invitrogen, 12-0114-82), FITC anti-mouse F4/80 (Invitrogen, 11-4801-82), and PE anti-CD206 antibodies (Biolegend, 141706). To detect Intracellular IFNγ, cells were fixed with 4% paraformaldehyde (Sigma, F8775) for 15 min and were permeabilized with 0.2% Triton X-100 (Sigma, T9284) for 10 min and were probed with FITC anti-mouse IFNγ (Biolegend, 505805). Data were acquired on a FACSCantoII (BD Biosciences) and were analyzed using FACSDiva software.
7
Immune Cell Profiling in Lung and Peritoneal Samples
8
Flow Cytometry Analysis of BMDC Activation
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BMDCs of each treatment group in experiment (1) were collected and were divided into two tubes, to which anti-mouse MHCII-PE-(yanin5-conjugated) Cy5, anti-mouse CD80-FITC, and anti-mouse CD11c-PE (phycoerythrin), anti-mouse CD86-FITC, and anti-CD40-PE-cy5 (eBioscience), respectively, were added, followed by the addition of 500 μl of 2% PFA in PBS, pH 7·4. A blank control was included. All samples were incubated in the dark at 4°C for 30 min, and were then subjected to flow-cytometry detection of surface molecules of BMDCs. Data were collected for at least 1×104 cells, and the experiment was carried out four times.
9
Multiparametric Flow Cytometry Immunophenotyping
10
Immune Cell Profiling in Lung and Peritoneal Samples
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BAL cells were labeled for anti-mouse CD11c-phycoerythrin ((PE), clone HL3, BD Biosciences) and anti-mouse F4/80-allophycocyanin ((APC), clone BM8, BioLegend) antibodies. Peritoneal cells were labelled for anti-mouse F4/80-APC and anti-mouse CD11b-PE-Cy7 (clone M1/70, eBioscience). The FcR Blocking Reagent (Miltenyi Biotec) was used to prevent non-specific bonding of antibody conjugates. To discriminate live and dead cells, the eBioscience™ Fixable Viability Dye eFluor™ 506 (Thermo Fischer Scientific) was used based on the manufacturer’s recommendation. The CD11c-F4/80 double positive alveolar macrophages, F4/80loCD11blo small peritoneal macrophages, and F4/80hiCD11bhi large peritoneal macrophages were sorted by FACSAria™ III (BD Biosciences). Approximately 15,000–25,000 cells were separated for transcript analysis. The flow cytometry analysis and cell sorting were performed by BD FACSAria III (BD Biosciences) using BD FACSDiva Software 6.0 (BD Biosciences).
To determine total immune cell numbers of BAL samples, the CountBright™ Absolute Counting Beads (Thermo Fischer Scientific), Fc Receptor blocker (Miltenyi Biotec), anti-mouse F4/80-APC, anti-mouse CD11c-PE, and anti-mouse CD24-fluorescein-5-isothiocyanate (FITC) (clone M1/69, eBioscience) were used. The acquired flow cytometry data were analyzed with FlowJo v10.8 (BD Biosciences).
To determine total immune cell numbers of BAL samples, the CountBright™ Absolute Counting Beads (Thermo Fischer Scientific), Fc Receptor blocker (Miltenyi Biotec), anti-mouse F4/80-APC, anti-mouse CD11c-PE, and anti-mouse CD24-fluorescein-5-isothiocyanate (FITC) (clone M1/69, eBioscience) were used. The acquired flow cytometry data were analyzed with FlowJo v10.8 (BD Biosciences).
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