Anti-human CD4-PE (cat 300508) was from BioLegend. Anti-human CD83-FITC (cat 556910), anti-human HLA-ABC-PE (cat 555553), anti-human HLA-DR-PE (cat 555812), anti-human CD80-FITC (cat 557226), anti-human CD86-APC (cat 555660), anti-human CD25-PE (cat 555432), anti-human CD127-FITC (cat 561697), Anti-human CD4-PE-Cy7 (BD) (cat 348809), anti-TNF-PE-Cy7 (cat 557647), anti-human IL-2-PE (cat 559334), anti-human CD3-APC-H7 (cat 560176), anti-human CD4-CF594 (cat 5562281), anti-human CD4-PB (pacific blue), anti-human CD8-BV605 (cat 564116), anti-human CD8-PerCP-Cy5.5 (cat 341050), anti-human IFN-γ–AF700 (cat 557995), anti-human CD35-PE-Cy7 (cat 557741), anti-human IL-10 (cat 554707) were from Becton Dickinson, Pharmigen. Anti-human Foxp3-APC (cat 17-4776-41) was from eBioscience. Anti-human CD14-PE (cat A07764) was from Beckman Coulter. Anti-human CD45RO-APC/Cy7 (cat 304227), anti-human CD45RA-PE (cat 304205), anti-human CD3-APC (cat 300411), and anti-human CD40-APC (cat 313008) were from BioLegend). Anti-human IL-10-BV421(cat 501421), anti-human CD45RA-BV711 (cat 304137), anti-human CD127-BV711 (cat 351327) from Biolegend, anti-human CD45RO-ECD (cat IM2712) (BC), anti-human FoxP3-PE (cat 12-4777-42), from eBiosciences.
Anti human hla dr pe
Manufactured by BD
Sourced in United States
Anti-human HLA-DR-PE is a flow cytometry reagent that binds to the HLA-DR antigen expressed on the surface of human cells. It is conjugated with the fluorescent dye phycoerythrin (PE) for detection and analysis by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti human cd34 pe, by BD (2 mentions)
Fitc anti human cd90, by BD (2 mentions)
Percp cy5.5 anti human cd 105, by BD (2 mentions)
Apc anti human cd 73, by BD (2 mentions)
Anti human cd45 pe, by BD (2 mentions)
Anti human foxp3 pe, by Thermo Fisher Scientific (1 mentions)
Pe anti human cd4, by BioLegend (1 mentions)
Anti human hla abc pe, by BD (1 mentions)
Pe anti human cd25, by BD (1 mentions)
Anti human cd4 pe cy7, by BD (1 mentions)
Anti tnf pe cy7, by BD (1 mentions)
Anti human foxp3 apc, by Thermo Fisher Scientific (1 mentions)
Anti human cd40 apc, by BioLegend (1 mentions)
Anti human il 10 bv421, by BioLegend (1 mentions)
Anti human cd3 apc, by BioLegend (1 mentions)
Facscelesta flow cytometer, by BD (1 mentions)
Anti ifnγ af488, by BD (1 mentions)
Anti foxp3 af488, by BioLegend (1 mentions)
Facscalibur cytometer, by BD (1 mentions)
Anti human cd83 apc, by BD (1 mentions)
6 protocols using anti human hla dr pe
1
Comprehensive Immunophenotyping Protocol
2
Phenotyping Macrophage Subsets via Flow
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To detect M1/M2 macrophages, the collected macrophages were incubated for 30 min at room temperature with the following specific antibodies: anti-human HLA-DR-PE (BD, 555,812) and anti-human CD206-APC (BD, 550,889). After three washes, the cells were incubated with a fixation/permeabilization working solution (eBioscience, 00-5523-00) in the dark for 60 min at room temperature. After another three washes, the cells were incubated with an anti-human CD68-FITC antibody (BD, 562,117) in the dark for 30 min. Then, the cells were resuspended in 200 µl of PBS and collected into tubes. All samples were analyzed using a BD FACSCelesta flow cytometer.
3
Detailed Immunophenotyping of Dendritic Cells
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Primary unconjugated antibodies used in this study were anti-human CD1a clone OKT6 culture supernatant (from American Type Culture Collection), anti-human HLA-I clone W6/32, anti-human DC-SIGN (27 (link)) (a gift from Dr. Angel Corbí). ICAM-I IgG supernatant (HV5/3) and VCAM-I supernatant, a gift from Dr. Sánchez-Madrid. Fluorescein-coupled polyclonal goat anti-mouse IgG (H + L; Caltag Laboratories) was used as the secondary antibody in FACS analysis. The following conjugated antibodies were used: anti-human CD1d-PE, anti-human HLA-DR-PE, anti-human CD80-PE, anti-human CD86-FITC, anti-human CD25-APC, anti-human CD83-APC (all from BD Biosciences), anti-human iNKT-PE (Miltenyi Biotec), and anti-human CD8-PECy7 (Beckman Coulter), anti IFNγ-AF488 (BD Biosciences), anti FoxP3-AF488 (Biolegend). Flow cytometry was performed following standard protocols on a BD FACSCalibur cytometer. Graph bars summarizing flow cytometry data are expressed as relative mean fluorescence intensity (MFI), which is defined as the MFI of any sample divided by the MFI of the immature dendritic cells (iDCs).
For intracellular staining, cells were fixed with PBS-PFA 4% for 20 min, then, the cells were washed twice in PBS containing 0.1% saponin (Sigma-Aldrich), incubated with the desired antibodies in 100 ml of PBS containing 1% saponin for 30 min at room temperature, and washed with PBS/0.1% saponin buffer.
For intracellular staining, cells were fixed with PBS-PFA 4% for 20 min, then, the cells were washed twice in PBS containing 0.1% saponin (Sigma-Aldrich), incubated with the desired antibodies in 100 ml of PBS containing 1% saponin for 30 min at room temperature, and washed with PBS/0.1% saponin buffer.
4
Intracellular ROS Probe and Antibody Assay
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Intracellular nonspecific reactive oxygen species (ROS) probe H2DCFDA
and 10% H2O2 were obtained from Sigma. RPMI 1640 culture
medium and FBS were purchased from Gibco. Anti-human-CD3-FITC,
anti-human-CD62L-PE, and anti-human-HLA-DR-PE Abs were obtained from BD
Biosciences.
and 10% H2O2 were obtained from Sigma. RPMI 1640 culture
medium and FBS were purchased from Gibco. Anti-human-CD3-FITC,
anti-human-CD62L-PE, and anti-human-HLA-DR-PE Abs were obtained from BD
Biosciences.
5
Characterization of hWJ-MSCs by Flow Cytometry
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The hWJ-MSCs that were used were obtained from passage 3 at 80% confluence. The cells were detached from cell culture flasks (Falcon®, Coring, Inc., New York, NY, USA) using trypsin/ethylenediaminetetraacetic acid (EDTA) solution 0.05% (GibcoTM Thermo Fisher Inc., Waltham, MA, USA) and washed with PBS. Then, the hWJ-MSCs were incubated for 30 min at room temperature with saturating concentrations of the following monoclonal antibodies labeled with fluorochromes: PerCP/Cy5.5 anti-human CD 105, FITC anti-human CD90, APC anti-human CD 73, PE anti-human CD 34, PE anti-human CD 45, PE anti-human CD19, PE anti-human CD 11b, PE/Cy7 anti-human HLA-ABC, and PE anti-human HLA-DR (all purchased from BD Biosciences (San Jose, CA, USA). After removing the excess antibody through 3 washes with PBS, the hWJ-MSCs were ana-lyzed according to standard protocols using a flow cytometer (FACS Calibur™, BD Biosciences, San Jose, CA, USA) and FlowJo software (Becton, Dickinson & Company, Franklin, NJ, USA). The cell population was identified, and whether cells were positive or negative for each marker was determined based on the criteria of the International Society for Cell and Genetic Therapy (ISCT).
6
Isolation and Characterization of MSCs
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In this experimental study, MSCs serum-free medium
was ordered from Shanghai Pumao Biotechnology
(Shanghai, China), and trypsin and Dulbecco’s phosphate
buffered saline (DPBS) were ordered from Gibco (Grand
Island, NY, USA). The antibodies of APC-anti-human
CD73, FITC-anti-human CD90, PerCP-Cy5.5- antihuman CD105, PE-anti-human CD34, PE-anti-human
CD45, and PE-anti-human HLA-DR were purchased
from BD Pharmingen (NJ, USA). The CD63 and β-actin
antibodies were ordered from Absin Bioscience (Shanghai,
China) and Cell Signaling Technology (Danvers, MA,
USA), respectively.
was ordered from Shanghai Pumao Biotechnology
(Shanghai, China), and trypsin and Dulbecco’s phosphate
buffered saline (DPBS) were ordered from Gibco (Grand
Island, NY, USA). The antibodies of APC-anti-human
CD73, FITC-anti-human CD90, PerCP-Cy5.5- antihuman CD105, PE-anti-human CD34, PE-anti-human
CD45, and PE-anti-human HLA-DR were purchased
from BD Pharmingen (NJ, USA). The CD63 and β-actin
antibodies were ordered from Absin Bioscience (Shanghai,
China) and Cell Signaling Technology (Danvers, MA,
USA), respectively.
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