INK128, rapamycin, and metformin were purchased from Selleckchem Inc. Recombinant mouse IFN-α2, anti-CD11b-fluorescein isothiocyanate (FITC), anti-Gr1-allophycocyanin (APC), anti-Ly6G-phycoerythrin (PE), and anti-Ly6C-APC were purchased from Biolegend Inc. TRIzol reagent and SYBR green dye were purchased from Invitrogen Inc. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco Inc. Collagenase type D and DNase I were purchased from Roche Inc. Pristane, N-methyl-2-pyrrolidone (NMP), and polyvinyl pyrrolidone (PVP) were purchased from Sigma Inc. Antibodies for α-tubulin (2144), p-S6 (4858S), S6 (2217S), p-4EBP-1 (2855S), 4EBP-1 (9644T), p-mTOR (5536T), p-AMPK (25375), and IRF-8 (83413T) were purchased from Signal Technology Inc. MDSC isolation kit, recombinant mouse IL-6, and GM-CSF were purchased from Miltenyi Biotec Inc. R848, CpG, poly I:C, and TNF-ɑ were purchased from Enzo Life Science Inc. Mouse albumin enzyme linked immunosorbent assay (ELISA) quantitation set, mouse anti-IgG, and anti-dsDNA IgG kit were purchased from Bethyl Laboratories Inc.
Anti ly6c apc
Manufactured by BioLegend
Sourced in United States
Anti-Ly6C-APC is a monoclonal antibody that binds to the Ly6C antigen, which is expressed on a subset of mouse myeloid cells. The antibody is conjugated with the fluorophore APC, allowing for the detection and analysis of Ly6C-positive cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11b pe cy7, by BioLegend (4 mentions)
Anti cd45 percp cy5, by BioLegend (2 mentions)
Anti f4 80 pe, by Bio-Rad (2 mentions)
Facsdiva software, by BD (2 mentions)
Anti cd11b efluor450, by Thermo Fisher Scientific (2 mentions)
Anti cd206 apc, by BioLegend (2 mentions)
Anti cd11b pe, by BioLegend (2 mentions)
Facscanto 2, by BD (2 mentions)
Metformin, by Selleck Chemicals (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Mdsc isolation kit, by Miltenyi Biotec (1 mentions)
Sybr green dye, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Selleck Chemicals (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Recombinant mouse il 6, by Miltenyi Biotec (1 mentions)
Poly 1 c, by Enzo Life Sciences (1 mentions)
Ink128, by Selleck Chemicals (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Polyvinylpyrrolidone, by Merck Group (1 mentions)
N methyl 2 pyrrolidone, by Merck Group (1 mentions)
9 protocols using anti ly6c apc
1
Synergistic Immunomodulatory Therapy Assessment
2
Cardiac infiltrating immune cells
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C57BL/6 and B1R−/− mice were infected with Dm28c TCTs (106 parasites; i.p.). At 15 dpi, the mice were euthanized and the cardiac tissue was processed. The hearts were perfused with 10 mL of PBS after a cut to the superior vena cava, and then cut into small pieces and digested in solution containing DNAse (0.1 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) and collagenase (1 mg/mL; Sigma-Aldrich) for 1 h at 37 °C. For monocytes/neutrophils analysis, Fc receptors were blocked with anti-CD16/CD32 (BD Biosciences, New York, NY, USA), cells were stained with anti-CD11b-PECy7 (Biolegend, San Diego, CA, USA), anti-Gr1-FITC BD Biosciences, anti-Ly6C-APC (Biolegend), and anti-F4/80-PE antibodies (Biolegend). Cells were acquired with a FACSCalibur (BD Biosciences, New York, NY, USA) and the data were processed with Summit software (DAKO, Colorado, Inc., Fort Collins, CO, USA). Flow cytometry analyses were performed at Plataforma de Imuno-Análise (PIA, IBCCF, UFRJ, Brazil), Centro Multiusuário Darcy Fontoura de Almeida (CMDFA, IBCCF, UFRJ, Brazil), IBCCF/UFRJ.
3
Multiparametric Flow Cytometry of Skin Immune Cells
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Cell suspension from lesional skin was incubated with antibody mixture including anti-F4/80-PE (BioRad, Munich, Germany), anti-Ly6G-FITC, anti-CD11b-PECy7, anti-CD45-PerCPCy5.5 and anti-Ly6C-APC (all Biolegend) for 30 min at 4 °C. After washing, cells were stained with Zombie NIR™ (Biolegend) to label dead cells and immediately analyzed. Flow cytometry was performed with BD FACS Canto II (BD Biosciences, Heidelberg, Germany) and data analyzed using BD FACSDIVATM software (BD Biosciences). Cells were gated as described [14 (link)]. In brief, single cells were selected and dead cells excluded via positive stain for Zombie NIR™. Within the population of viable cells, co-expression of CD45 and CD11b was used to mark immune cells. Within the population of CD45+/CD11b+ cells PMN are defined by expression of Ly6G. Cells negative for Ly6G are further defined as monocytes/macrophages (Mo/Ma) due to their Ly6C and F4/80 expression.
4
Pristane-Induced Cell Profiling
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Pristane was injected i.p. in WT and CRAMP−/− mice and cells were collected via peritoneal lavage with PBS or after erythrocyte lysis from peripheral blood at day 0 and 7 after injection. Cells were stained extracellularly with anti-CD11b-eFluor450 (eBioscience), anti-Ly6C-APC (BioLegend), anti-CD115-PE (BioLegend), and anti-Ly6G-APC (BioLegend) to determine cell types. Intracellular CRAMP staining was performed with an anti-CRAMP (Innovagen) antibody labelled with an Alexa Fluor 488 antibody labelling kit (LifeTechnologies). Cells were then analyzed by cytofluorometry on a Gallios machine (Beckman Coulter).
5
Multiparametric Flow Cytometry of Myeloid Cells
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Cells were incubated with antibody mixture including anti-F4/80-PE (BioRad), anti-Ly6G-FITC, anti-CD11b-PECy7, anti-CD45-PerCPCy5.5 and anti-Ly6C-APC or, anti-CD301-APC or anti-CD206 APC (Biolegend, San Diego, CA, USA) for 15 min and subsequently, 0.4 µl zombie dye (Biolegend) was added for 5 min. For detection of S100A9 protein intracellular staining was performed using the True-Nuclear™ Transcription Factor Buffer Set. Cells were first stained with antibody mixture including anti-CD45-PerCPCy5.5, anti-CD11b-PECy7, anti-Ly6G-APC, anti-F4/80-PE or anti-CD45-PerCPCy5.5, CD31-PE, CD90-PE-Cy7 for 15 min, washed and fixed for 45 min before intracellular staining with goat anti-S100A9 antibody (R&D) for 30 min, washing and addition of secondary antibody (anti-goat Alex 488, Life Technology) for further 30 min.
Flow cytometry was performed with BD FACSCantoTMII or BD FACSLyric™ and data analysed with BD FACSDivaTM Software or BD FACSuite™ Application (BD, Heidelberg, Germany). Using forward and sideward scatter single cells were gated. Living singlets were chosen from zombie-negative cells as described 31 (link).
Flow cytometry was performed with BD FACSCantoTMII or BD FACSLyric™ and data analysed with BD FACSDivaTM Software or BD FACSuite™ Application (BD, Heidelberg, Germany). Using forward and sideward scatter single cells were gated. Living singlets were chosen from zombie-negative cells as described 31 (link).
6
Multicolor Flow Cytometry for MSCs and MDSCs
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The following antibodies were used for the MSC-2 cells: anti-CD11b-FITC, anti-Ly6-C-APC and anti-Ly6-G-PE, all purchased from Biolegend. For patient-derived MDSCs, we used anti-CD33-APC, anti-CD11b-AP, and anti-HLA-DR-PECy7, purchased from Beckman Coulter. Data were acquired using an LSRII flow cytometer (BD Biosciences). All colors were evaluated against their respective isotype controls and samples with no staining.
7
Multiparametric Flow Cytometry of Cardiac Cells
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After treatment of isolated cardiac cells with mouse TruStain FcX (BL-101320, BioLegend), samples were stained with anti-F4/80-APCfireTM (BL-123108, BioLegend), anti-CD11b-PE (BL-101208, BioLegend), anti-Ly6C-APC (BL-128016, BioLegend), anti-Ly6C-FITC (BL-128006, BioLegend), and/or anti-GPR68 (CSB-PA060199; CUSABIO). Cells stained with anti-GPR68 were further stained with anti-rabbit IgG-AF647 (ab-150107, Abcam). To detect IL-6- and TNFα-expressing cells, cells were also incubated with 10 μg/mL of Brefeldin A (Funakoshi), followed by fixation with 2% paraformaldehyde and permeabilization with Intracellular Staining Permeabilization (BioLegend). After fixation and permeabilization, cells were stained for anti-IL-6-APC (BL-504507, BioLegend) and anti-TNF-α-APC (BL-506307, BioLegend). Dead cells were labeled by eFluor 780 viability dye (BD-565388, BD Biosciences, Erembodegem, Belgium). Stained cells were applied to flow cytometry (Aria III; BD Biosciences) and the results were analyzed using FlowJo (Tree Star).
8
Fcγ receptor expression on mouse immune cells
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Blood cells of naïve BALB/c WT and Ncf1** mice were analyzed for expression of different Fcγ receptors in steady state or after 2 h of incubation with propidium iodide-labelled SNECs. After hypotonic water lysis cells were stained with anti-CD11b-eFluor450 (eBioscience) and anti-Ly6C-APC (BioLegend), and either anti-CD64-PerCP/Cy5.5, anti-CD16/32-PE/Cy7, or anti-CD16.2-PE (all from BioLegend). Analysis was carried out using a Beckman Coulter Gallios™.
9
Flow Cytometric Analysis of Immune Cell Subsets
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The following fluorescent-labeled monoclonal antibodies and staining reagents are used according to the manufacturer's specifications: anti-APC-CY7-live (BioLegend), anti-BV510-CD45 (BioLegend), anti-F4/80-BV421 (BioLegend), anti-CD11B-PE CY7 (BioLegend), anti-CD206-APC (BioLegend), anti-CD80-percp/Cy5.5 (BioLegend), anti-LY6C-APC (BioLegend), anti-CD11B-PE (BioLegend), anti-LY6G-PE CY7 (BioLegend), anti-CD3-PE CY7 (BioLegend), anti-CD4-BV421 (BioLegend), anti-CD8-percp/CY5.5 (BioLegend). The cells were proceeded by FACS Calibur flow cytometry and analyzed by the FlowJo software.
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