The following antibodies were used for flow cytometry: anti-CD4 (RM4-4), anti-CD11c (N418), anti-CD86 (GL1), anti-CD40 (1C10), anti- IFN-γ (XMG1.2), anti-IL-4 (BVD6-24G2), anti-IL-17 (eBIo17B7: all from eBioscience), anti-CD25 (3C7 from BD), anti-Foxp3 (150D from BioLegend), and anti-CD11b (M1/70 from BioLegend). For cytokine staining, sample cells were stimulated in culture medium containing10 ng/ml PMA and 1 ng/ml Iono (6 h), with 3 μg/ml brefildin A (eBioscience 00-4506) for the last 2 h, then treated with the Intracellular Fixation & Permeabilization Buffer Set (eBioscience 88-8824-00), according to the manufacturer’s protocol. For Foxp3 staining, samples were treated with the Foxp3 buffer set (BioLegend 421403). All samples were measured on a BD FACSVerse analyzer, and data were analyzed using Flowjo software.
Foxp3 buffer set
Manufactured by BioLegend
The Foxp3 buffer set is a collection of buffers designed for the detection and analysis of Foxp3, a transcription factor that is a key regulator of regulatory T cells (Tregs). The set includes a fixation/permeabilization concentrate, a fixation/permeabilization diluent, and a permeabilization buffer, which are used to prepare cells for intracellular staining of Foxp3.
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Multi-Parametric Flow Cytometry Analysis
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Multi-Parameter Flow Cytometry Analysis
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Fluorochrome-conjugated antibodies against mouse CD3, CD4, CD8, CD19 and Ki-67, and against human Ki-67, Zombie-Green/Violet fixable viability dyes and Foxp3 buffer set were purchased from Biolegend (San Diego, CA). Lymphocytes were stained for cell surface markers.
Live and dead cells were distinguished by staining the cells with Zombie dyes or 7-AAD. For Ki-67 staining, cells were fixed with CytoFix/CytoPerm buffer, washed with PBS plus 1% FBS and 1x CytoPerm buffer, and incubated with antibodies against mouse or human Ki-67. Jurkat and Raji cells were stained with Zombie-Green dye followed by fixation, permealization and staining with anti-human Ki-67. (Biolegend, San Diego, CA). For Annexin V staining, cells were washed twice with plain PBS and once with Annexin V binging buffer (10mM HEPES, pH7.4, 140mM NaCl, 2.5mM CaCl 2 ) and incubated with fluorochrome-conjugated Annexin V (Biolegend) in Annexin V binding buffer for 15min at room temperature. The cells were washed twice in Annexin V binding buffer, and re-suspended in Annexin V binding buffer. 7-AAD was added to the cells before analyzed by flow cytometry to distinguish dead and live cells.
Live and dead cells were distinguished by staining the cells with Zombie dyes or 7-AAD. For Ki-67 staining, cells were fixed with CytoFix/CytoPerm buffer, washed with PBS plus 1% FBS and 1x CytoPerm buffer, and incubated with antibodies against mouse or human Ki-67. Jurkat and Raji cells were stained with Zombie-Green dye followed by fixation, permealization and staining with anti-human Ki-67. (Biolegend, San Diego, CA). For Annexin V staining, cells were washed twice with plain PBS and once with Annexin V binging buffer (10mM HEPES, pH7.4, 140mM NaCl, 2.5mM CaCl 2 ) and incubated with fluorochrome-conjugated Annexin V (Biolegend) in Annexin V binding buffer for 15min at room temperature. The cells were washed twice in Annexin V binding buffer, and re-suspended in Annexin V binding buffer. 7-AAD was added to the cells before analyzed by flow cytometry to distinguish dead and live cells.
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