Py230 cells were cultured either alone or in co-culture with MSC2 cells for 24 hours under normal conditions. The cells were then trypsinized and re-suspended in PBS at concentration of 1×106 cells/ml. Cells were then labeled with PE-conjugated E-cadherin antibody and APC-conjugated Anti-CD11b antibody (Biolegend, San-Diego,CA ) as per the manufacturer’s recommendation. The cells were then incubated at 4°C for 30 minutes in the dark. After washing the cells, the fluorescence was read/processed using a Becton Dickinson FACSAria III.
Apc conjugated anti cd11b antibody
Manufactured by BioLegend
APC-conjugated anti-CD11b antibody is a laboratory reagent that binds to the CD11b cell surface antigen. CD11b is a marker expressed on myeloid cells, including monocytes, macrophages, and granulocytes. The APC fluorescent dye is conjugated to the antibody, allowing for detection and analysis of CD11b-positive cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 3, by BD (1 mentions)
Anti mouse cd16 cd32 antibody, by BD (1 mentions)
Apc cy7 conjugated anti mouse cd45 antibody, by BioLegend (1 mentions)
Canto 2, by BD (1 mentions)
Ficoll gradient, by GE Healthcare (1 mentions)
Apc conjugated cd4 antibody, by BioLegend (1 mentions)
Cellquest software, by BD (1 mentions)
Pe conjugated anti foxp3 antibody, by BioLegend (1 mentions)
Foxp3 intracellular staining kit, by BioLegend (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
3 protocols using apc conjugated anti cd11b antibody
1
Flow Cytometry Analysis of Cell Surface Markers
2
Isolation and Characterization of Osteoclast Precursors
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To analyze the frequency of OCP subsets, BM cells were collected from the mice, and washed three times with PBS supplemented with 4% FBS. After washing, the cells were treated with an anti-mouse CD16/CD32 antibody (BD, San Jose, CA) for 15 min on ice to block non-specific binding. The cells were then stained with APC-Cy7-conjugated anti-mouse CD45 antibody (Biolegend, San Diego, CA), FITC-conjugated anti-mouse CD117 antibody (Biolegend, San Diego, CA), PE-conjugated anti-mouse CD115 antibody (Biolegend, San Diego, CA), or APC-conjugated anti-CD11b antibody (Biolegend, San Diego, CA) for 30 min on ice. The cells were washed with PBS with 4% FBS, and analyzed using a Canto II (BD Biosciences, San Jose, CA) analyzer and Flowjo software to process the data (Tree Star, Ashland, OR). Monocytes/macrophages obtained from mouse bone marrow were cultured with M-CSF (30 ng/ml) for 3 days and treated with LPS (10 ng/ml) for 2 more days and were collected to analyze the RANK expression. APC-conjugated anti-mouse CD265 (RANK) antibody (Biolegend, San Diego, CA) was used to observe the RANK expression in OCPs.
3
Quantification of T-reg and M2-monocytes in PBMCs
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Peripheral blood mononuclear cells (PBMCs) were isolated from 8 ml of peripheral blood (PB) using a ficoll gradient (GE healthcare, Buckinghamshire, UK). For analysis of regulatory T cells (T reg s) in PBMCs, the FoxP3 Intracellular Staining Kit (BioLegend, San Diego, CA, USA) was used; 2 x10 7 PBMCs were fixed and then incubated with PE-conjugated anti-FoxP3 antibody and APC-conjugated CD4 antibody (BioLegend, San Diego, CA, USA) for 30 min at 4 °C in the dark. To analyze the M2-skewed monocyte phenotype in PBMCs, 1 x10 7 PBMCs were incubated with an APC-conjugated anti-CD11b antibody (BioLegend) and then with a FITC-conjugated anti-CD206 antibody (Abcam) for 30 min at 4 °C, in the dark. After washing twice with FACS buffer, the proportion of T reg s and M2-phenotype monocytes in PBMCs was analyzed using a FACSCalibur flow cytometer and the CELLQuest software (Becton Dickinson, San Jose, CA, USA).
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