Brain tissue slices from Tg2576 mice were examined for CD11b and CD11c immunoreactivity in proximity to amyloid plaques in the cortex by ring analysis, as previously described (Frautschy et al., 2001 (link); Frautschy et al., 1998 (link); Lim et al., 2001 (link)). Briefly, brain sections were taken at 2.5 mm posterior to Bregma ( ± 0.3 mm). Sections were double labeled for Aβ (anti-Aβ 1–13 DAE; blue) and microglia (anti-mouse CD11c (BD Biosciences)) with avidin biotin-peroxidase kit and the peroxidase detector (diaminobenzidine; brown). For acquisition of histological and immunohistochemical images for analysis, the blue slice (filter) of the RGB stack was chosen and density slice threshold selected such that the blue plaque would not be picked up when imaging the brown microglia; all images were captured using the same density slice threshold. Images were acquired at 20×, digitized, and quantitatively analyzed with NIH-Image public domain software. A custom Pascal macro subroutine measured the microglia as a % of total area, in relation to distance from the Aβ-immunoreactive plaque center in each of a series of three concentric circles within and around Aβ-immunoreactive plaques, the ring widths of which correspond to the radius of the Aβ plaque, where Ring 1 defines 2 plaque radius, and Rings 2 and 3 are one and two plaque radii away from the plaque edge.
Anti mouse cd11c
Manufactured by BD
Anti-mouse CD11c is a monoclonal antibody that binds to the CD11c antigen, which is expressed on the surface of dendritic cells and some other myeloid cells in mice. This antibody can be used for the identification and isolation of CD11c-positive cells in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b, by BioLegend (2 mentions)
Mhcii, by BD (1 mentions)
Facs verse flow cytometer, by Tree Star (1 mentions)
Flowjov software, by Tree Star (1 mentions)
Golgiplug, by BD (1 mentions)
Cd8 antibody, by BD (1 mentions)
Anti ifn γ, by BD (1 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
Anti il 4, by BD (1 mentions)
Anti mouse gr 1, by BD (1 mentions)
Anti mouse mhc 2, by BioLegend (1 mentions)
Anti mouse cd45, by BD (1 mentions)
Fc blocking anti mouse cd 16 32 antibody, by BD (1 mentions)
Efluor 450, by Thermo Fisher Scientific (1 mentions)
Cfse dye, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by BioLegend (1 mentions)
Tnf α, by BioLegend (1 mentions)
Anti mouse pd 1, by BioXCell (1 mentions)
Ifn γ, by BioLegend (1 mentions)
Mhcii, by Thermo Fisher Scientific (1 mentions)
9 protocols using anti mouse cd11c
1
Quantifying Microglial Responses to Amyloid Plaques
2
Analyzing Mouse Immune Cell Phenotypes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse BMDCs or splenocytes were washed with PBS. For surface marker extracellular staining, the cells were incubated with anti-mouse CD11c (557,400, BD Biosciences), CD80 (560,526, BD Biosciences), CD86 (552,692, BD Biosciences), MHC-II (562,367, BD Biosciences), CD11b (101,211, Biolegend), CD4 (553,046, BD Biosciences), or CD8 antibodies (551,162, BD Biosciences) at 4 °C for 30 min. For intracellular cytokine staining, the splenocytes were stimulated with DnaJ (10 μg/ml) for 8 h in presence of Golgi plug™ (51-2301KZ, BD Bioscience). The splenocytes were then treated for surface markers (CD4 or CD8), fixed/permeabilised with a Cytofix/Cytoperm solution (51-2090KZ, BD Bioscience) and then stained with anti-IFN-γ (557,735, BD Biosciences) and anti-IL-4 (554,435, BD Biosciences) antibodies at 20–25 °C for 30 min. All events were acquired on a FACSverse flow cytometer and analysed using the FlowJoV software (Tree Star).
To determine cytokine (IFN-γ, TNF-α, IL-1β, IL-10, IL-17a, MCP-1, IL-1α, and IL-6) concentrations in the uterine tissue, multi-analyte flow assay kits (740,446, Biolegend, San Diego, CA, USA) were used as indicated by the manufacturer.
To determine cytokine (IFN-γ, TNF-α, IL-1β, IL-10, IL-17a, MCP-1, IL-1α, and IL-6) concentrations in the uterine tissue, multi-analyte flow assay kits (740,446, Biolegend, San Diego, CA, USA) were used as indicated by the manufacturer.
3
Eosinophil Quantification in BALF Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected from bronchoalveolar lavage fluid (BALF) and pre-incubated with Fc-blocking anti-mouse CD16/32 antibody (BD Pharmingen, #553,141) before the staining process. Subsequently, dead cells were excluded by DAPI staining (BD Pharmingen, #564,907). The cells then underwent surface staining with anti-mouse CD45 (BD Pharmingen, #561,037), anti-mouse CD11b (BD Pharmingen, #561,688), anti-mouse GR-1 (BD Pharmingen, #561,103), anti-mouse CD-11c (BD Pharmingen, #561,022), and anti-mouse MHCII (Biolegend, #107,613) for 30 min at 4 °C. All samples were analyzed using Cytek Dxp Athena flow cytometer, and the data were processed using FlowJo software (version 10). The method for eosinophil count was conducted following established protocols from previous study [23 (link)], and is illustrated in Fig. S1 . Initially, live leukocytes were selected based on the expression of CD45 and exclusion of DAPI, effectively removing debris, erythrocytes, and dead cells. Subsequently, lymphocytes were differentiated through the analysis of the SSC-A/CD11b plot, while neutrophils were identified using the SSC-A/GR1 plot among the diverse cell populations. Ultimately, eosinophils were isolated from the remaining cells using the MHC-II/CD11c plot.
4
Tumor Cell-BMDC Interaction Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded into 6-well plates (3 × 105 cells per well) overnight and then treated with 60 μM of mitomycin C for 30 min. Cell death was analyzed 48 h later via flow cytometry for calreticulin (ADI-SPA-601PE-F, enzo life sciences) expression, and supernatants were used to detect HMGB1 via an ELISA (6010, Chondrex, Woodinville, WA, USA), respectively.
Phagocytosis was performed by coculturing inactivated tumor cells with BMDCs. Tumor cells were stained with 2.5 μM of CFSE dye (C34573, Invitrogen, Waltham, MA, USA), and seeded in 48-well plates. BMDCs were stained with 1 μM of efluor 450 (65–0842-85, eBioscience). BMDCs were cocultured with tumor cells in a ratio of 1:1 or 1:3 and incubated for 2 h at 37 °C. BMDCs were collected for the flow cytometry analysis of phagocytosis.
BMDC maturation was assessed after coculturing with MMC-treated or untreated tumor cells. After 24 h, BMDCs were stained with anti-mouse CD11c and anti-mouse CD80 (BD Biosciences), and then analyzed via flow cytometry.
Phagocytosis was performed by coculturing inactivated tumor cells with BMDCs. Tumor cells were stained with 2.5 μM of CFSE dye (C34573, Invitrogen, Waltham, MA, USA), and seeded in 48-well plates. BMDCs were stained with 1 μM of efluor 450 (65–0842-85, eBioscience). BMDCs were cocultured with tumor cells in a ratio of 1:1 or 1:3 and incubated for 2 h at 37 °C. BMDCs were collected for the flow cytometry analysis of phagocytosis.
BMDC maturation was assessed after coculturing with MMC-treated or untreated tumor cells. After 24 h, BMDCs were stained with anti-mouse CD11c and anti-mouse CD80 (BD Biosciences), and then analyzed via flow cytometry.
5
CFSE-based Lymphocyte Proliferation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We cultured CT-26 cells in culture plates for 24 hours. The cells were subjected to varying doses of IR, and the plates were incubated for 0, 24, 48, and 72 hours. Cells of each group were harvested and washed thrice with PBS, followed by staining with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) at a final concentration of 5 μM. The labeled cells were washed thrice with PBS. DCs were harvested and stained with anti-mouse CD11c (BD Pharmingen) at 4ºC for 30 minutes, followed by co-culture with irradiated CT-26 cells in a 96-well plate (200 μL/well) for 2 hours. The cells were analyzed with FC500.
6
Formulation and Characterization of CGMP R-DOTAP Liposomal Nanoparticles
7
Dendritic Cell Maturation and Chemotaxis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DCs used in this study were isolated from mouse bone marrow (BMDCs) and cultured as described previously (34, 35). Briefly, bone marrow cells from femur were obtained by flushing femur with PBS supplemented with 2% heat inactivated FBS. These cells were then centrifuged and resuspended in Tris-ammonium chloride to lyse RBCs. The cells were spun and then filtered with a 70μm filter and were resuspended in RPMI-1640 supplemented with antibiotic and heat inactivated 10% FBS containing recombinant mouse GM-CSF (20ng/ml) and IL-4 (5ng/ml). Loosely adherent dendritic cells were collected and re-plated on day 7 in fresh RPMI medium and were harvested on day 8 and subjected to chemotaxis assay.
To analyse maturation, 0.5x10 6 cells/well in a 24 well-plate were left untreated or treated with 10μg/ml each of ECP, EDN and HPR in separate wells for 48 hours at 37ºC. After 48 hours, cells were stained with anti-mouse CD11c (BD Biosciences), CD80 (BD Biosciences), CD86 (eBioscience), CD40 (eBioscience) and MHCII (eBioscience), and analysed on FACS Canto II.
To analyse maturation, 0.5x10 6 cells/well in a 24 well-plate were left untreated or treated with 10μg/ml each of ECP, EDN and HPR in separate wells for 48 hours at 37ºC. After 48 hours, cells were stained with anti-mouse CD11c (BD Biosciences), CD80 (BD Biosciences), CD86 (eBioscience), CD40 (eBioscience) and MHCII (eBioscience), and analysed on FACS Canto II.
8
Multicolor Flow Cytometry of Immune Cells
9
Immunofluorescent Labeling of Mouse Eye Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse eyes were enucleated and embedded in Tissue-Tek OCT compound, and frozen in liquid nitrogen. 10 micrometer-thick sections were cut and mounted to polylysine-coated glass slides. After a 10-min fixation in 4% paraformaldehyde, slides were blocked with 10 mM sodium phosphate buffer containing 2% BSA for 1 hour at room temperature. Sections were then incubated with mouse anti-CD11c (BD Biosciences) and anti-collagen IV (EMD Millipore, MA). This was followed by a secondary antibody, FITC -conjugated goat anti-hamster and cy3- conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories 1:100), and slides were mounted with Vectorshield mounting medium containing 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) mounting media and examined under confocal microscopy. Controls were similarly treated, but the primary antibody was replaced with IgG.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!