Mouse bone marrow-derived DCs (1 × 107 cells/mL in a 96-well flat-bottom culture plate) were treated with Alexa 488-labeled K-type CpG ODNs (1.25 µg CpG ODNs/mL) or CA nanoparticles containing Alexa 488-labeled K-type CpG ODNs (CA-Alexa 488-CpG; 1.25 µg CpG ODNs/mL) for 10, 30, 90, 120, or 180 min. Cells were stained with 0.4 w/v% trypan blue solution (Wako, Osaka, Japan) to quench any Alexa 488-labeled K-type CpG ODNs bound to the cell surface, and then the cells were analyzed by means of flow cytometry (NovoCyte Flow Cytometer, ACEA Bioscience, San Diego, CA, USA). To separate various subsets of DCs from the collected cells, we incubated the cells with anti-mouse CD16/CD32 antibody (TONBO biosciences, San Diego, CA, USA), anti-mouse B220 antibody (BioLegend), anti-CD11c antibody (BioLegend), and anti-CD11b antibody (BioLegend) in the absence of trypan blue. In this way, the DCs were separated into the following subsets: B220+ CD11c+ plasmacytoid DCs, CD11b+ CD11c+ conventional DCs, and CD11b− CD11c+ conventional DCs.
Anti cd11b antibody
Manufactured by BioLegend
Sourced in United States
The Anti-CD11b antibody is a monoclonal antibody that binds to the CD11b cell surface antigen. CD11b is a subunit of the integrin Mac-1 (CD11b/CD18), which is expressed on the surface of various immune cells, including monocytes, macrophages, and neutrophils. This antibody can be used for the identification and characterization of cells expressing CD11b.
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Lab products found in correlation
Anti cd11c antibody, by BioLegend (3 mentions)
Novocyte flow cytometer, by Agilent Technologies (2 mentions)
Anti cd49b antibody, by BioLegend (2 mentions)
Anti ly6g antibody, by BioLegend (2 mentions)
Anti cd45 antibody, by BioLegend (2 mentions)
Anti cd19 antibody, by BioLegend (2 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Human ab serum, by Merck Group (1 mentions)
Anti icam 1 antibody, by BioLegend (1 mentions)
Anti pd l1 antibody, by BioLegend (1 mentions)
Isotype matched antibody, by BioLegend (1 mentions)
Isotype matched control antibody, by BioLegend (1 mentions)
Facsverse, by BD (1 mentions)
Facsuite, by BD (1 mentions)
Human fcr blocking reagent, by BioLegend (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Endofree plasmid mega kit, by Qiagen (1 mentions)
Anti ifn γ antibody, by BD (1 mentions)
Anti f4 80 antibody, by BioLegend (1 mentions)
Anti cd45 antibody, by BD (1 mentions)
16 protocols using anti cd11b antibody
1
Characterization of DC Subsets Uptake of CpG ODNs
2
Neutrophil-Infected Red Blood Cell Interaction
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Primary neutrophils or differentiated PLB985 cells were incubated with fluorescent late‐stage iRBC either expressing GFP or stained using MitoTracker as described, at a 10:1 ratio at 37°C for different time periods. Samples were washed, and the extent of neutrophils–iRBC interaction (% fluorescent neutrophils) was determined using flow cytometry. Opsonization of iRBCs was performed by culturing iRBCs with AB human serum (Sigma) for 30 min at 37°C. To assess ligand–receptor specificity to this interaction, we performed these assays using anti‐Cd11b antibody (Biolegend Cat # 101211, 10 μl/ml) and a non‐PfEMP1‐blocking anti‐ICAM‐1 antibody (Biolegend Cat # 322702, 10 μl/ml) as negative controls. An anti‐ICAM‐1 monoclonal antibody (15.2) that blocks the PfEMP1‐binding site (Thermofisher, MA180910, 10 μl/ml) was used as blocking antibody as described (Baratin et al, 2007 (link)). All antibodies were incubated with iRBCs for 30 min at room temperature in culture media prior to flow cytometry interaction assays. To inhibit phagocytosis, neutrophils were pre‐incubated with 5 µM cytochalasin D (Sigma‐Aldrich), an actin polymerization inhibitor, for 1 h at 37°C prior to the addition of fluorescent iRBCs.
3
PD-L1 Expression Analysis in Macrophages and Tumors
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Human monocyte-derived macrophages were treated with human FcR-blocking reagent (BioLegend) and then reacted with phycoerythrin-labeled or Alexa 488-labeled anti-human PD-L1 antibody (BioLegend) or isotype-matched control antibody (BioLegend). For analysis of murine subcutaneous tumor, anti-CD11b antibody, anti-PD-L1 antibody, and isotype-matched antibodies (BioLegend) were used. The stained cell samples were analyzed on a FACSverse (Becton Dickinson, Franklin Lake, NJ, USA) flow cytometer with FACSuite (Becton Dickinson) software.
4
Assessing Purity of Isolated Mouse Peritoneal Macrophages
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We performed the flow cytometry to assess the purity of isolated mouse peritoneal macrophages according to a previous study [18 (link)], i.e., isolated peritoneal macrophages were washed twice with PBS and then added with or without 1 μl each of monoclonal anti-F4/80 antibody (eBioscience, San Diego, CA, USA), anti-PE antibody (Thermo- Fisher, Waltham, MA, USA), and anti-CD11b antibody (Biolegend, San Diego, CA, USA) and incubated at 4oC for 30 min. The cells were then subjected to flow cytometric analysis (BD FACSria II, BD Biosciences, San Jose, CA, USA).
5
Nanoparticle-based Immunostimulant Delivery
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Folate (FA), monomethoxyl poly (ethylene glycol) (MPEG), ε-CL and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) were purchased from Sigma–Aldrich (Darmstadt, Germany). 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) was purchased from Avanti Polar Lipids (AL, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were both purchased from Gibco (NY, USA). The antibodies used in this study were summarized: rat anti-mouse CD31 polyclonal antibody, rat anti-mouse Ki67 polyclonal antibody, rat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Servicebio, Wuhan, China); The anti-CD4 antibody, anti-CD8 antibody, anti-CD69 antibody, anti-CD11c antibody, anti-CD80 antibody, anti-CD86 antibody, anti-MHCII antiobody, anti-CD45 antibody, anti-CD11b antibody and anti-IFN-γ antibody (BD Pharmingen, NJ, USA); The anti-CD11b antibody, anti-CD206 antibody, anti-F480 antibody, anti-CD107a antibody, anti-CD49b antibody, anti-CD274 (PDL-1) antibody (Biolegend, CA, USA). The pvax and pIL-12 were extracted and purified using a QIAGEN Endofree Plasmid Mega Kit (Hulsterweg, Netherlands). The contents of murine IFN-γ, TNF-α and IL-12 p70 were detected by corresponding ELISA kits from Invitrogen (CA, USA).7-AAD/Annexin-V apoptosis kit was purchased from BD, Pharmingen (CA, USA).
6
Alpinetin Modulates Neuroinflammation Pathways
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Alpinetin (CAS. 36052‐37‐6, purity ≥98%) was purchased from Chemstrong Scientific Co., Ltd. Cy3‐labeled goat anti‐rat IgG, Alexa Fluor 555‐tagged donkey antirabbit/mouse IgG, Alexa Fluor 488‐tagged goat antirabbit/mouse IgG, horseradish peroxidase‐labeled secondary antibodies, antifluorescence quenching agent (containing DAPI), and CCK‐8 reagent were purchased from Beyotime. Lipopolysaccharide (LPS) was purchased from Merck. Anti‐COX‐2, anti‐Iba‐1, anti‐CD68, anti‐GFAP, and anti‐GAPDH antibodies were purchased from Abcam. Primary antibodies against iNOS, p‐JAK2, p‐STAT3, STAT3, β‐actin, Bcl‐xL, GFAP, NeuN, GAP43, and MAP2 were obtained from Cell Signaling Technology. Alexa Fluor 488 phalloidin was also from CST. The anti‐CD11b antibody was bought from Biolegend. The anti‐JAK2 antibody was provided by Affinity. WP1066 purchased from GlpBio. Transwell chamber was purchased from Corning (0.4 μm pores). Calcein AM/propidium iodide (PI) was bought from Solarbio. The JC‐1 MMP assay kit and the ROS fluorescence test kit were from Elabscience. The polymerase chain reaction (PCR) primers were synthesized by Servicebio while the kit for RNA extraction came from EZBioscience. The reagents for quantitative real‐time PCR (qPCR) were purchased from Vazyme.
7
Isolation and Flow Cytometry of Cochlear Immune Cells
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After both sides of the temporal bones were isolated from the mice, the bony capsule of the cochlea was carefully removed in PBS. The whole cochlea tissue was extracted from the cochlear bony capsule and temporal bone. After the cochlear tissue was transferred to a new dish, the tissue was trypsinized for 10 min, followed by gentle grinding on a 40 μm filter. The cells were stained with dye-conjugated anti-CD11b antibody (Biolegend, 1:200) and anti-Ly6G antibody (Biolegend, 1:200) for 30 min at 4 °C. Flow cytometry was performed using BD LSR II (Becton Dickinson, Franklin Lakes, NJ, USA). The analysis was performed using the FlowJo software (Becton Dickinson).
8
Quantitative Analysis of Kidney Inflammation
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To quantitatively analyze the type of inflammatory cells infiltrated into the AA kidney, kidneys were enzymatically digested to prepare the cell suspensions [27] (link), followed by the analysis of flow cytometry (FACSCanto II flow cytometer BD Biosciences, Co., Tokyo, Japan). Briefly, kidneys were dissected, placed in Hank's Balanced Salt Solution (HBSS) containing 1.6 mg/ml collagenase D for 30 min at 37 °C, and then washed twice in HBSS. Following the erythrocyte lysis with lysis buffer (BD Pharm Lyse, BD Biosciences Ltd., Rockville, MD), cells were resuspended in PBS containing 2 vol% FBS and 0.1 vol% sodium azide (FACS buffer). The kidney cells suspension was blocked by anti-CD16/32 antibody (BioLegend Ltd., San Diego, CA), washed, and stained with an anti-CD11b antibody (BioLegend Ltd., San Diego, CA), an anti-CD 45 antibody against CD45 expressed on the surface of mouse macrophages (BioLegend Ltd., San Diego, CA) in FACS buffer. 7-Amino-Actinomycin D (7-AAD, BD Biosciences Ltd., Rockville, MD) was used to distinguish dead cells from viable cells. The immunostained cells were analyzed on FACS, and the analysis was performed by the BD FACSDiva software (v6.13 BD FACSDiva software, BD Biosciences Ltd., Rockville, MD).
9
Isolation of Murine Macrophages and Neutrophils
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The femurs and tibias of C57 BL/6 J mice were removed, placed in 75% ethanol (5 min) and then washed using Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Germany). Cells within the bone marrow were prepared as a single-cell suspension. For macrophage isolation, cells within the bone marrow were cultured in DMEM with 10% (v/v) fetal bovine serum and recombinant murine macrophage colony-stimulating factor (20 ng/mL, Novoprotein, China). On the seventh day, bone marrow-derived macrophages (BMDMs) were harvested. For neutrophil isolation, cells were isolated from the single-cell bone marrow suspension using Percoll density gradient centrifugation and separated via positive selection for CD11b+Ly6G+ cells (Anti-CD11b antibody, BioLegend, USA, 101205; Anti- Ly6G antibody, Tonbo Biosciences, 20-5931) on flow cytometry (BD Influx, USA).
10
Visualization of NHE and Cell Membrane
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Immunofluorescence stainings for visualization of inflammasomes were described in our previous publication31 (link). For visualization of NHE and cell membrane, BMDMs were stimulated with NHE component CWT or Cmut. or C + B for 1 h, or C + B + A for 30 min, washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min, followed by blocking in 1% BSA in PBS for 1 h. Cells were incubated with a rabbit sera anti-NHE-C (1:200 dilution in 1% BSA)55 (link), a mouse anti-NHE-B, or -A (1:200 dilution in 1% BSA)55 (link), an mouse anti-Histidine antibody (1:200 dilution in 1% BSA, ab18184, Abcam), and a rat fluorescein isothiocyanate-conjugated anti-CD11b antibody (1:200 dilution in 1% BSA, 101205, BioLegend) overnight at 4 ° C. PBS containing 0.05% Tween-20 was used to wash between incubation steps. An anti-mouse secondary Rhodamine red antibody (115295146, Jackson ImmunoResearch) or rabbit secondary Rhodamine red antibody (111295144, Jackson ImmunoResearch) were used. The samples were mounted with VECTASHIELD Hardset Mounting Medium with DAPI (H-1500, Vector Laboratories, Inc.) and analyzed using a Leica SP5 confocal microscope.
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