DMEM and RPMI 1640 cell culture media, antibiotics, and nonessential amino acids were purchased from Life Technologies (Grand Island, NY). CD14+ monocytes were isolated by MACS CD14 microbeads from Miltenyi Biotec (Auburn, CA). Human Abs, including CD16 allophycocyanin, CD16 FITC, CD14 FITC, CD40 FITC, and CD86 FITC, were purchased from eBioscience (San Diego, CA). Abs CD14 allophycocyanin, CD14 PE, CD163 PE, CD11c allophycocyanin, CD68 PE, CD206 allophycocyanin, DC-SIGN FITC, and isotype control Abs were purchased from BD Pharmingen (Franklin Lakes, NJ). Phospho-p44/42-ERK1/2 and anti-mouse IgG PE were obtained from Cell Signaling Technology (Danvers, MA). Human IL-10 Ab and mouse IgG1 isotype control were from R&D Systems (Minneapolis, MN). miR-27a inhibitor, mimic, and scrambled controls were purchased from Ambion Life Technologies (Carlsbad, CA). Lipofectamine RNAiMAX transfection reagent was from Life Technologies. The sprouty2 construct was obtained from OriGene (Rockville, MD), which was transfected by Roche (Indianapolis, IN) X-tremeGENE transfection reagent. ERK inhibitor, U0126, was procured from EMD Millipore (Billerica, MA).
Cd68 pe
Manufactured by BD
Sourced in United States
CD68-PE is a fluorochrome-conjugated antibody that specifically binds to the CD68 antigen, a glycoprotein expressed on the lysosomal membrane of macrophages and monocytes. This product is intended for use in flow cytometry and other immunoassays to identify and characterize cells of the monocyte-macrophage lineage.
Automatically generated - may contain errors
Lab products found in correlation
Cd163 pe, by BD (4 mentions)
Cd86 fitc, by Thermo Fisher Scientific (2 mentions)
Cd16 fitc, by Thermo Fisher Scientific (2 mentions)
Cd40 fitc, by Thermo Fisher Scientific (2 mentions)
Dc sign fitc, by BD (2 mentions)
Cd14 fitc, by Thermo Fisher Scientific (2 mentions)
Cd14 pe, by BD (2 mentions)
Cd45 pe, by BD (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Cd40 pe cy7, by BD (2 mentions)
Cd11c apc, by BD (2 mentions)
Macs cd14 microbeads, by Miltenyi Biotec (1 mentions)
Dmem and rpmi 1640 cell culture media, by Thermo Fisher Scientific (1 mentions)
Cd14 allophycocyanin, by BD (1 mentions)
Cd11c allophycocyanin, by BD (1 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
X tremegene transfection reagent, by Roche (1 mentions)
U0126, by Merck Group (1 mentions)
Cd31 pe, by BD (1 mentions)
Cd83 pe, by BD (1 mentions)
8 protocols using cd68 pe
1
Monocyte Isolation and Characterization
2
Flow Cytometry Analysis of CD34+ Cells
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Human nonexpanded and expanded PB-CD34+ cells (n = 5) were analyzed by flow cytometry (FACSCalibur flow cytometer, (Becton-Dickinson (BD), San Jose, CA). Dead cells were excluded by propidium iodide (PI) staining (Sigma, St Louis, MO). The CD34+ cells were incubated with a FcR blocking reagent (Miltenyi Biotec, Auburn, CA) and incubated with the monoclonal antibodies for 30 minutes at 4 °C. The stained cells were washed, resuspended, and then analyzed using Quad Statistics of CellQuest software (BD). The following monoclonal antihuman antibodies were used to characterized the CD34+ cell population: CD34-FITC (BD), CD31-PE (BD), CD133-PE (Miltenyi Biotec, Auburn, CA), CD68-PE (BD), CD83-PE (BD), VE-cadherin-PE (BD), VEGFR-2-PE (R&D Systems, Minneapolis, MN), Tie-2 (BD), CD117-PE (BD), CD45-PE (BD), IgG2a-FITC isotope controls (Miltenyi Biotec), and IgG1-PE isotope controls (Miltenyi Biotec).27 (link)The DNA content analysis was assessed by staining ethanol-fixed cells with PI and monitoring with the FACSCalibur flow cytometer. At least 20,000 cells were collected and analyzed with CellQuest software. Cell cycle distributions were calculated with ModFit LT cell-cycle analysis software (Verity Software House, Topsham, ME).
3
Investigating Immune Cell Responses to TLR Agonists
4
Stem Cell Immunophenotyping Protocol
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ASCs were characterized by immunostaining with differently fluorescent labeled antibodies for mesenchymal and hematopoietic stem cell markers: CD90-APC, CD49b-APC, CD44-FITC, CD105-PE/APC, CCR5-APC, CD4-eFluor, CD34-PE, CD14-PE-Cy5, CD45-PE and CD68-PE (BD Biosciences, Franklin Lakes, NJ) (26 (link)). Analogously TZM-bl and Jurkat-T-cells were stained for CCR5 (CD195, BD Bioscience, Cat. #556903) surface expression using standard staining methods. If positive and negative cells were not distinguishable as two separate populations, Overton histogram subtraction technique was utilized for determining the fraction of positive cells (64 (link)).
5
Quantifying Macrophage Subpopulations in Bone Marrow
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Bone marrow samples were obtained by standard bone marrow puncture using sterile heparin anticoagulant tubes. Bone marrow samples were filtered using flow cytometry tubes. CD14-FITC (Cat No.: 555397), CD68-PE (Cat No.: 565595), CD64-APC (Cat No.: 561189), CD40-PEcy7 (Cat No.: 561215), CD206-PE (Cat No.: 555954), CD163-PEcy7 (Cat No.: 556018), and isotype control antibodies (BD Biosciences, USA) were added to the tubes. The samples were then stained for 15 min in the dark at room temperature. After red blood cell lysis, the cells were washed with PBS. Finally, the cells were detected using a FACSCalibur flow cytometer (BD Biosciences, USA). Data analysis was performed using the Cell Quest software (Becton Dickinson, version 3.1).
Macrophages were defined as CD14+CD68+ cells. M1 macrophages were defined as CD64+CD40+ macrophages. M2 macrophages were defined as CD206+CD163+ macrophages (detail in Supplemental Figure1 ).
Macrophages were defined as CD14+CD68+ cells. M1 macrophages were defined as CD64+CD40+ macrophages. M2 macrophages were defined as CD206+CD163+ macrophages (detail in Supplemental Figure
6
Single-cell analysis of mouse lung cells
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Single cell suspensions from whole mouse lungs were prepared using the lung dissociation kit (Miltenyi Biotec, Auburn, CA) as per the manufacturer's instructions. Cells were stained with fluorescently labeled antibodies directed against PD-L1, PD-L2 (InVivoMAb; BioXcell, West Lebanon, NH), CC10, SPC (Santa Cruz Biotechnology, Santa Cruz, CA), CD3, CD68-PE, CD11b-FITC (BD Pharmingen, Billerica, MA, USA), PD-L1-PE-cy7, and F4/80-FITC (Biolegend, Dedham, MA). Flow cytometry data was collected using the BD FACSAriaIIIu and analyzed with FlowJo V10 software.
7
Peripheral Blood Immune Cell Profiling
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The absolute counts of total lymphocytes, monocytes, and neutrophils in the peripheral blood of all subjects were measured by flow cytometry with a Guava easyCyte 8 system (Merck Millipore, USA). Cell-surface monocyte phenotypic analysis was performed after staining with human anti-CD14 1 (link) and anti-CD16 1 (link). Fluorochrome-conjugated monoclonal antibodies allophycocyanin (APC)-CD40, phycoerythrin (PE)-CD86, APC-HLA-DR, PE-CD11b, APC-CD11c, APC-CD62L, PE-CD68, PE-CD4, peridinin chlorophyll protein complex (PerCP)-CD3, PE-CD45RO, APC-CCR7, fluorescein isothiocyanate (FITC)-CD40L, and PE-CD163 were purchased from BD Biosciences Pharmingen (Franklin Lanes, NJ, USA).
8
Multicolor Flow Cytometry for Macrophages
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