The extracted mLNs were treated with 3 ml of a collagenase solution (collagenase from Clostridium histolyticum, type VIII; 0.75 mg/ml) and incubated for 10 min at 37°C. After this incubation and centrifugation, the supernatant was discarded, while the cell pellets were resuspended and separated through a cell sieve (70 μm). Afterwards the immune cell subset composition was analyzed by flow cytometry. The Following antibodies were used: CD3‐APC‐Cy7 (clone 145‐2C11), CD103‐PE (clone 2E7), MHCII‐PE‐Cy7 (clone M5/114.15.2) (all acquired from Biolegend); B220‐VioBlue® (clone RA3‐6B2, acquired from Miltenyi); and CD11c‐APC (clone HL3, BD Biosciences). Cytokine and FOXP3 staining was performed after surface staining with CD90‐APC‐Cy7 (clone 30‐H12), CD4‐FITC (clone GK15), CD25‐APC (clone PC61) (all acquired from Biolegend). For intracellular stainings, cells were washed twice and treated with a True‐Nuclear™ Transcription Factor Buffer set (BioLegend) according to the manufacturer's instructions, including incubation with FOXP3‐Pacific blue (clone MF‐14, BioLegend). Flow cytometric analysis was performed using a flow cytometer (Gallios™, Beckmann Coulter) and Kaluza Analysis 1.3 software (Beckmann Coulter).
Foxp3 pacific blue clone mf 14
Manufactured by BioLegend
FOXP3-Pacific blue (clone MF-14) is a fluorochrome-conjugated antibody that targets the FOXP3 protein. FOXP3 is a transcription factor that is commonly used as a marker for regulatory T cells. The Pacific blue fluorochrome is used to label the antibody, enabling detection and analysis by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Cd11c apc clone hl3, by BD (1 mentions)
True nuclear transcription factor buffer set, by BioLegend (1 mentions)
Kaluza analysis 1, by Beckman Coulter (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Pharm lyse, by BD (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Ova323 339 peptide, by InvivoGen (1 mentions)
Cd8 apc cy7, by Thermo Fisher Scientific (1 mentions)
Ova257 264 peptide, by InvivoGen (1 mentions)
2 protocols using foxp3 pacific blue clone mf 14
1
Isolation and Characterization of Immune Cells from Murine Lymph Nodes
2
Multiparameter Flow Cytometry Analysis
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CD3e-APC or CD3e-PE (clone 145-2C11), CD4-APC/Cy7 or CD4-PerCp (clone GK1.5), CD8α-APC/Cy7 or CD8α-PacBlue (clone 53–6.7), CD11c-APC or CD11c-PE (clone N418), CD11b-PerCP (clone M1/70), CD19-PerCP/Cy5.5 (clone HIB19), CCR4-APC or CCR4-PE/Cy7 (clone 2G12), CD25-PE (clone PC61), IFN-γ-Pacific Blue (clone XMG1.2), and Foxp3-Pacific Blue (clone MF-14) were all from BioLegend. T regs were stained with a Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. For intracellular IFN-γ staining, peripheral blood or splenocytes were incubated with red blood cell lysis buffer (BD Pharm Lyse; BD Bioscience) for 3 min. Lymphocytes were then stimulated with 100 nM OVA257–264 peptide or OVA323–339 peptide (both InvivoGen) for 1 h at 37°C before 1 µg/ml brefeldin A (Sigma-Aldrich) was added. After 3 h, cells were surface stained with CD8-APC/Cy7 and CD19-PerCP, then fixed and permeabilized using the Foxp3 Staining Buffer Set (eBioscience) and incubated with an anti–IFN-γ-Pacific Blue antibody. Pentamer staining was performed with H-2Kb OVA257–264 R-PE pentamers (ProImmune) according to the manufacturer’s instructions. Events were measured on a FACS Canto II flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar).
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