All antibody staining was preceded by 15 min of 1:50 FcγR block in FC buffer, on ice. Extracellular antibodies were then added to FC buffer containing FcγR block, and incubated for 45 min on ice. Intracellular staining was accomplished after surface staining using the Foxp3 staining kit (eBioscience). Myeloid subsets were stained with anti-CD1lb-FITC, anti-Ly6C-AF700, anti-MR-PE-Cy7, anti-CD11c-PE/Dazzle594, anti-Ly6G-BV605 (BioLegend), anti-MHCII-eFluor450 (eBioscience), and anti-F4/80-APC (BioRad). To evaluate Tregs, cells were stained with anti-CD3-AF488, anti-CD4-APC, anti-CD25-PerCP-Cy5.5 (BioLegend), and anti-Foxp3-PE (BD Pharmingen). To assess T cell activation, total tumor cells were incubated for 4 hr at 37°C in a 5% CO2 incubator with Protein Transport Inhibitor Cocktail containing brefeldin A and monensin, or with Cell Stimulation Cocktail containing protein transport inhibitors and PMA/ionomycin (eBioscience). Cells were then stained with anti-CD3-AF488, anti-CD4-PE, and anti-CD8-PerCP-Cy5.5 followed by intracellular stain with anti-IFNγ-APC (BioLegend). In addition, 1E5 CD3+ cells were stimulated with 4E4 CD3/CD28 Dynabeads (ThermoFisher) for 3 days, followed by staining using anti-CD3-PerCP-Cy5.5, anti-CD8-BV650, anti-CD4-BV605, anti-IFNγ-APC, anti-TNFα-BV421 (BioLegend), and anti-GranzymeB-PE (eBioscience).
Anti f4 80 apc
Manufactured by Bio-Rad
The Anti-F4/80-APC is a fluorescently labeled antibody that binds to the F4/80 antigen. The F4/80 antigen is a cell surface glycoprotein expressed on macrophages and monocytes. The APC (Allophycocyanin) fluorescent label allows for the detection and analysis of cells expressing the F4/80 antigen using flow cytometry.
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Lab products found in correlation
Anti ifn γ apc, by BioLegend (2 mentions)
Anti foxp3 pe, by BD (2 mentions)
Anti cd4 pe, by BioLegend (2 mentions)
Anti cd4 bv605, by BioLegend (2 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (2 mentions)
Anti cd3 af488, by BioLegend (2 mentions)
Anti mhcii efluor450, by Thermo Fisher Scientific (2 mentions)
Anti cd25 percp cy5, by BioLegend (2 mentions)
Anti ly6c af700, by BioLegend (2 mentions)
Anti mr pe cy7, by BioLegend (2 mentions)
Anti cd11c pe dazzle594, by BioLegend (2 mentions)
Anti ly6g bv605, by BioLegend (2 mentions)
Anti granzyme b pe, by Thermo Fisher Scientific (1 mentions)
Anti cd4 apc, by BioLegend (1 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions)
Anti cd8 percp cy5, by BioLegend (1 mentions)
Pma ionomycin, by Thermo Fisher Scientific (1 mentions)
Anti tnfα bv421, by BioLegend (1 mentions)
Anti cd8 bv650, by BioLegend (1 mentions)
Anti cd3 percp cy5, by BioLegend (1 mentions)
5 protocols using anti f4 80 apc
1
Comprehensive Immune Cell Profiling
2
Multiparametric Flow Cytometry of Immune Cells
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Tumors were dissociated as for cell inoculation and subjected to flow cytometry (FC) analysis, gating on live cells lacking staining with Live/Dead Aqua (ThermoFischer). All antibody staining was preceded by FcγR block on ice (eBioscience/Fisher). Extracellular antibodies were then added and samples were incubated on ice. Intracellular staining was accomplished after surface staining using the FoxP3 staining kit (eBioscience). To evaluate myeloid subsets, total cells were stained with anti-CD11b-FITC, anti-CD45-BV650, anti-Ly6C-AF700, anti-MR-PE-Cy7, anti-CD11c-PE/Dazzle594, and anti-Ly6G-BV605 (BioLegend); anti-MHCII-eFluor450 (eBioscience); anti-CD86-PE (Miltenyi); and anti-F4/80-APC (BioRad). T cells were enumerated by staining with anti-CD45-AF700, anti-CD3-AF488, anti-CD4-PE, anti-CD8-BV655, and anti-CD69-APC-Cy7 (BioLegend), followed by intracellular stain with anti-IFNγ-APC (BioLegend). To evaluate Tregs, total cells were stained with anti-CD3-AF488, anti-CD4-BV605, and anti-CD25-PerCP-Cy5.5 (BioLegend), and then stained intracellularly with anti-FoxP3-PE (BD Pharmingen).
3
Multiparametric Flow Cytometry Analysis
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Isolation of PEC was described elsewhere (7 (link), 11 (link)). For FACS analyses, cells were stained for 20 min at 4°C with the following fluorochrome-labeled monoclonal antibodies: anti-CD11c-allophycocyanin (APC), anti-B220-PE (both from BD PharMingen), anti-CD11b-Pacific Blue (from Caltag/Invitrogen), and anti-F4/80-APC (AbD Serotec). Cells were washed and analyzed with a LSRII flow cytometer (Becton Dickinson). Analyses were performed using BD FACSDiva™ 8.0.1 and FlowJo® 7.6.5 and 10.7.1.
4
Flow Cytometric Immunophenotyping of Leukocytes
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Before staining, cells were incubated with FcBlock reagent (anti-CD16/CD32, Biolegend, Ozyme-France) to block the FcγRII/III receptors. The cellular content of the peritoneal exudates was characterized using a combination of fluorochrome conjugated mAbs: CD45-vioblue (REA737, Miltenyi Biotec), Ly6G-FITC (1A8, Miltenyi Biotec) F4/80-APC (CI:A3-1; AbD Serotec, BioRad, Luxembourg), Ly6C-PE (HK1.4, Biolegend), and CCR3-APC-Fire (J073E5, Biolegend). At analysis, doublets and dead cells, labeled with 7-AAD (Biolegend), were excluded. All samples were acquired using a MACS Analyzer (Miltenyi Biotec) flow cytometer and analyzed using FlowJo software (TreeStar, USA). The gating strategies are presented in Supplementary Material. Mammary gland cells were labeled with the following fluorochrome conjugated mAbs: anti-CD45-PECy7 (I3/2.3; Southern Biotech), Anti-Ly6G-FITC (1A8, Miltenyi Biotec), and anti-F4/80-APC (CI:A3-1; AbD Serotec) to identify leukocytes, neutrophils, and macrophages, respectively. At analysis, doublets, red blood cells, and lymphocytes were excluded based on CD45 staining. All samples were acquired using a CytoFLEX (Beckman Coulter, USA) flow cytometer and analyzed using CytExpert software (Beckman Coulter, USA).
5
Tumor Cell Isolation and Characterization
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Wild-type and CtsB−/−S−/− RT2 tumors were isolated and processed for fluorescence-activated cell sorting as previously described (Pyonteck et al. 2012 (link)) using the following antibodies: CD31-FITC (1:100; BD Pharmingen), CD45-PE (1:200; BD Pharmingen), anti-F4/80-APC (1:100; Serotec), and DAPI for dead cell exclusion. The cells were sorted on a fluorescence-activated cell sorting Aria flow cytometer (BD Biosciences), and fractions were collected: a mixed population of live cells (DAPI−), purified tumor cells (DAPI− CD31− CD45− F4/80−), and macrophages (DAPI− CD31− CD45+ F4/80+).
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