M-chlorin e6 PDT was performed as described above. Mice were euthanized by cervical dislocation on day 2 after LED irradiation. The tumors were removed and treated with 10% collagenase (Wako), shredded using a gentleMACS™ Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany), and passed through a 70-μm filter. Next, 1.0 × 106 cells were mixed with 1 μL of Fc-block/100 μL of stain buffer (BD Biosciences, San Jose, CA) and left on ice for 10 min. Subsequently, 1 μL of fluorescent antibody (CD11b-APC [BD Biosciences], CD206-BV421 [Biolegend, San Diego, CA], CD80-PE [BD Biosciences], CD86-PE-Cy7 [BD Biosciences], CD45-PE-Vio®770 [Miltenyi Biotec], CD3-FITC [Miltenyi Biotec], CD8a-APC-H7 [BD PharMingen, San Diego, CA], CD127-APC [BD PharMingen], CD25-PE [BD Biosciences], or CD4-BV421 [Biolegend]) diluted with 100 μL of stain buffer was added and left on ice for 30 min. The cells were then washed with 500 μL of phosphate-buffered saline (PBS) and suspended in 500 μL of stain buffer. Next, 5 μL of 7-AAD (BD Biosciences) was added to prepare a sampling solution, which was analyzed on a BD FACSVerse™ (BD Biosciences).
Cd127 apc
Manufactured by BD
Sourced in Canada, France, Germany
CD127-APC is a fluorescently-labeled antibody that specifically binds to the CD127 antigen, also known as the Interleukin-7 receptor alpha chain (IL-7Rα). CD127 is expressed on the surface of various immune cells, including T cells, B cells, and NK cells. The APC (Allophycocyanin) fluorescent dye is conjugated to the antibody, allowing for flow cytometric detection and analysis of CD127-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd86 pe cy7, by BD (1 mentions)
Cd206 bv421, by BioLegend (1 mentions)
Cd45 pe vio770, by Miltenyi Biotec (1 mentions)
Cd3 fitc, by Miltenyi Biotec (1 mentions)
Cd8a apc h7, by BD (1 mentions)
Cd4 bv421, by BioLegend (1 mentions)
Cd25 pe, by BD (1 mentions)
Cd80 pe, by BD (1 mentions)
Stain buffer, by BD (1 mentions)
7 aad, by BD (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Fc block, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Cd11b apc, by BD (1 mentions)
Cd11c pe cy7, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye efluor 450, by BioLegend (1 mentions)
Cd135 pe, by BD (1 mentions)
Fitc cd9, by BD (1 mentions)
Cd25 bv605, by BioLegend (1 mentions)
Lineage fitc, by BD (1 mentions)
5 protocols using cd127 apc
1
Tumor-Infiltrating Immune Cell Analysis
2
Comprehensive Immune Cell Profiling
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A panel of monoclonal antibodies was developed to enable phenotypic characterization of B and T lymphocyte subpopulations: CD3-FITC, CD4-APC, CD25-BV421, FoxP3-PE, CD19-PeCy7, CD9-BB700 (BD Bioscience, France), CD24-BV510, and CD38-APC-Cy7 (Sony Biotechnology, UK); hematopoietic stem and progenitor cells: CD34-BV421 (Biolegend, France), cKit-APC-H7, CD135-PE, lineage-FITC, Sca-1-BV510, CD16/32-PerCPCy5.5, CD127-APC (BD Bioscience), and TLR-4-PeCy7 (Biolegend); and dendritic cells: CD11c-PeCy7 (eBioscience, France), CMH-II-FITC, Siglec-H-BV510, CD11b-APC-H7, and CD103-PerCP5. For intracellular staining, cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences). For in vitro human B cell differentiation experiments, cells were stained with Fixable Viability Dye eFluor 450 to identify dead cells, followed by staining with CD25-BV605 (Biolegend), CD19-BUV395, CD9-FITC, CD27-BUV737, CD38-BV711 and intracellular IL-10-PE (BD Bioscience). Cells were analyzed on a Canto II flow cytometer (BD Biosciences). Data were acquired using Diva 8.0 software and analyzed with FlowJo X (TreeStar, Williamson Way, Ashland, USA). Fluorescence minus one staining controls were used for all panels, and dead cells were removed using viability staining.
3
Immunophenotyping of T-cell Subsets in HIV Infection
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Lymphocyte surface phenotypes were evaluated by flow cytometry on fresh peripheral blood of HIV-infected women alone, using fluorochrome-labeled antibodies: L/D-BV510 (Miltenyi Biotech, Germany); CD4-APC-H7, CD8-PE-Cy7, CD38-PE, CD45R0-PerCPCy5.5, CD45RA-PerCPCy5.5, CD127-APC, CD31-FITC, CCR7-APC, CD103-PE, CD95-FITC, CD69-FITC, PD1-PE (BD Biosciences, Palo Alto, CA).
T-cell subsets were defined as: naive CCR7+ CD45RA+, central memory CCR7+CD45RA−, effector memory CCR7− CD45RA−, and terminally differentiated CCR7− CD45RA+ subsets. Besides, we measured: activation (CD45R0/CD38/CD69), apoptosis (CD95), exhaustion (PD-1), IL7R (CD127), and Recent Thymic Emigrants (CD31 or CD103) on both CD4 and CD8 T-cell subsets.
Briefly, 1 × 106 peripheral blood mononuclear Cells were stained with the appropriate antibodies for 20 minutes at 4°C in the dark, washed and then acquired using FACSVerse cytometer (BD Biosciences, Palo Alto, CA).
T-cell subsets were defined as: naive CCR7+ CD45RA+, central memory CCR7+CD45RA−, effector memory CCR7− CD45RA−, and terminally differentiated CCR7− CD45RA+ subsets. Besides, we measured: activation (CD45R0/CD38/CD69), apoptosis (CD95), exhaustion (PD-1), IL7R (CD127), and Recent Thymic Emigrants (CD31 or CD103) on both CD4 and CD8 T-cell subsets.
Briefly, 1 × 106 peripheral blood mononuclear Cells were stained with the appropriate antibodies for 20 minutes at 4°C in the dark, washed and then acquired using FACSVerse cytometer (BD Biosciences, Palo Alto, CA).
4
Multiparameter Flow Cytometry Analysis
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Mouse anti‐human monoclonal antibodies against CD4‐PerCP, CD25‐FITC, CD127‐APC, CTLA‐4‐PE, CD11c‐PerCP Cy5.5, CD123‐APC 750, Lin‐APC, HLA‐DR‐FITC, and HLA‐DQ‐PE and mouse IgG1‐PE were purchased from BD Pharmingen and BECKMAN COULTER Inc. Red blood cell lysis buffer was purchased from BD Biosciences. The flow cytometer models were FACS‐Calibur (BD Biosciences) and CytoFLEX (BECKMAN COULTER). Analysis software FlowJo 7.6.1 (TreeStar) and CytExpert (BECKMAN COULTER) were used.
5
Evaluation of Spike Protein-Specific Immune Responses
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Spleens from untreated and immunized mice were passed through a 40-mm cell strainer to obtain a single-cell suspension. Spleen cells were cultured with spike protein (S: 1µg/ml), or medium only. After 72 h of incubation, IFN-γ and IL-17A concentrations were quantified in supernatants by ELISA.
For FACS analysis, spleen cells that were not re-stimulated with Spike protein were incubated with CD16/CD32 FcgRIII (1:100) to block IgG Fc receptors. Cells were treated with LIVE/DEAD Violet (Invitrogen), followed by surface staining with fluorochrome-conjugated anti-mouse Abs for various markers: CD3-Pe-Cy7 (BD), CD8-PE (BD), CD4-FITC (BD), CD44-PE (BD), CXCR5-PerCP-Cy5.5 (BD), CD127-APC (BD). Flow cytometry analysis was performed on an BD FACSAria Fusion. The results were analyzed using FlowJo software (TreeStar).
For FACS analysis, spleen cells that were not re-stimulated with Spike protein were incubated with CD16/CD32 FcgRIII (1:100) to block IgG Fc receptors. Cells were treated with LIVE/DEAD Violet (Invitrogen), followed by surface staining with fluorochrome-conjugated anti-mouse Abs for various markers: CD3-Pe-Cy7 (BD), CD8-PE (BD), CD4-FITC (BD), CD44-PE (BD), CXCR5-PerCP-Cy5.5 (BD), CD127-APC (BD). Flow cytometry analysis was performed on an BD FACSAria Fusion. The results were analyzed using FlowJo software (TreeStar).
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