Pooled splenocytes (2×105 cells/group, n = 3-4 / group) obtained from naive control, MPTP and calpeptin treated mice were stained with CD8-FITC (BD Biosciences, San Jose, CA) and CD4-PerCP (BD Biosciences, San Jose, CA) as described [20 (link)]. Flow cytometric two-parameter dot plots and quadrant statistics were generated using FACScan and CellQuest software (BD Biosciences, Mountain view, CA). For intracellular staining, cells were fixed and permeabilized using Fix and Perm reagents (BD Biosciences, San Jose, CA), and then incubated with specific antibodies [75 (link)]. Briefly, 5×105 cells per group were re-suspended in 0.5 mL of Fixation/Permeabilization Buffer (BD) and incubated at 2-8° C for 30 minutes. After washing, the cell pellet was re-suspended in Permeabilization/Wash Buffer and stained with Foxp3-APC (BD Biosciences, San Jose, CA). Cells were then analyzed on FACScan using CellQuest software as described above.
Foxp3 apc
Manufactured by BD
Sourced in United States
Foxp3-APC is a fluorescently labeled antibody that binds to the Foxp3 transcription factor. Foxp3 is a key regulator of the development and function of regulatory T cells. The APC (Allophycocyanin) fluorescent label allows for the detection and analysis of Foxp3-expressing cells using flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 percp, by BD (3 mentions)
Cd25 pe cy7, by BD (3 mentions)
Cd25 apc, by BD (3 mentions)
Cd8 fitc, by BD (2 mentions)
Cd62l fitc, by BD (2 mentions)
Cd4 pe, by BD (2 mentions)
Cd45 pe, by BD (2 mentions)
Cd11b apc cy7, by BD (2 mentions)
Cd4 fitc, by BD (2 mentions)
Cd4 pe cy7, by BD (2 mentions)
Cd19 fitc, by BD (2 mentions)
Cd25 pe, by BD (2 mentions)
Cd4 pacific blue, by BD (2 mentions)
Il 4 pe, by BD (2 mentions)
Cellquest, by BD (1 mentions)
Facscan, by BD (1 mentions)
Fix and perm reagent, by BD (1 mentions)
Permeabilization wash buffer, by BD (1 mentions)
Fixation permeabilization buffer, by BD (1 mentions)
Cd45ra fitc, by BD (1 mentions)
14 protocols using foxp3 apc
1
Multiparametric Flow Cytometry Analysis
2
Immunophenotyping of T Cell Subsets
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Samples were stained without further separation to minimize selective losses shortly after collection. Combinations of the directly conjugated monoclonal antibodies CD3-V500, CD4-PerCP, CD56-PE, CD127-BV421, CD25-PE-CY7, CD25-APC, CD62L-FITC, CD69-APC, CD45RA-FITC, CCR7-PE, Ki67-FITC, Foxp3-APC (BD Bioscience, Mountain View, CA, USA), CD16-APC/CY7, HLADR-APC/CY7 (Biolegend, San Diego, CA, USA) and their relative allotypes were used in individual 8-color flow cytometry assays to analyze the immunophenotype of regulatory T cells and effector T cells.
Intracellular staining was performed using the Intracellular Staining Kit (eBioscience, San Diego, CA, USA). The expression of Ki67 was determined in freshly isolated CD4+CD25highFoxp3+ regulatory T cells. The cellular secretion and function of cytokines were determined after incubation of cells for 5 h with phorbol myristate acetate (PMA) (100 ng/ml) plus ionomycin (2 ug/ml, all reagents from Sigma Chemical Co., St. Louis, MO, USA) to stimulate maximal production of IL-17 and IFN-γ; GolgiStop (0.7 μl/ml) was added to the samples during the last 4 hours to sequester the proteins in the cytoplasm [22 (link),23 (link)].
Intracellular staining was performed using the Intracellular Staining Kit (eBioscience, San Diego, CA, USA). The expression of Ki67 was determined in freshly isolated CD4+CD25highFoxp3+ regulatory T cells. The cellular secretion and function of cytokines were determined after incubation of cells for 5 h with phorbol myristate acetate (PMA) (100 ng/ml) plus ionomycin (2 ug/ml, all reagents from Sigma Chemical Co., St. Louis, MO, USA) to stimulate maximal production of IL-17 and IFN-γ; GolgiStop (0.7 μl/ml) was added to the samples during the last 4 hours to sequester the proteins in the cytoplasm [22 (link),23 (link)].
3
Quantification of Regulatory T Cells
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Peripheral blood of the patients was collected and lymphocytes were isolated with lymphocyte separation medium (TBD, China). After washing with PBS three times, CD4-PE and CD25-FITC (BD, USA) were added and incubated in the dark for 15 min, then washed with PBS three times. After breaking cell membrane, Foxp3-APC (BD, USA) was added and incubated in the dark for 15 min. After washing with PBS three times, the proportion of CD4+ CD25+ Foxp3+ Treg cells in lymphocytes was detected by flow cytometry (BD, USA). Results were recorded on a lymphocyte population gated according to forward scatter and side scatter characteristics (FSClow/SSClow) [15 (link)].
4
Flow Cytometry Analysis of T Cell Populations
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The following monoclonal antibodies were used for flow cytometry experiments: anti-CD3-APC, anti-CD8-PerCP, anti-CLA-FITC, anti-CD4-FITC, anti-CD25-PECY7, FOXP3-APC, purified anti-CD3 NA/LE and anti-CD28 NA/LE (anti-CD3/CD28), and FACSCalibur Flow Cytometer, all of which were obtained from BD Biosciences (Franklin Lakes, NJ, USA). CD8+T Cell Isolation Kit, anti-CLA MicroBead Kit, CD4+CD25+Regulatory T Cell Isolation Kit, 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), MS/LD Columns, and MiniMACS separator were purchased from Miltenyi Biotec (Cologne, Germany). Biotin-conjugated anti-transforming growth factor-β1 (anti-TGF-β1) or biotin-conjugated chicken IgG for a negative control were from Calbiochem (Darmstadt, Germany). RPMI 1640 medium, 100 U/mL penicillin G and 100 µg/mL streptomycin were purchased from Invitrogen (Carlsbad, CA, USA).
5
Flow Cytometry Analysis of Tumor Infiltrates
6
Multi-Dimensional Immune Profiling
7
Comprehensive Immune Cell Profiling
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Fleshly obtained hepatic nonparenchymal cells, spleen cells and Peripheral blood cells were stained with the following antibodies: 7-AAD, CD45-PE, CD45-FITC, CD3-FITC, CD3-APC, CD4-PE, CD8α-PE, CD25-APC, FoxP3-APC, CD11b-PE, F4/80-APC, NK1.1-PE, B220-APC, CD19-PE, GR-1-FITC (BD Biosciences, San Diego, CA). Then the cells were washed three times, resuspended in 500 µl of PBS. Data acquired by Accuri C6 (BD Biosciences) were analyzed using CFlow software (BD Biosciences).
8
Comprehensive Immune Profiling of TILs
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Flow cytometry analysis was performed by using a BD FACSVerse (BD Biosciences). Human TILs were stained with the following monoclonal antibodies: CD45-APC-H7, CD3ε-PerCP, CD8α-V500, CD56-PE-Cy7, CD19-FITC, IFN-γ-FITC, IL-17A-V450 (all from BD Pharmingen, Milan, Italy), IL-21-PE, T-bet-PE-Cy7 (clone 4B10), RORγt-APC (clone AFKJS-9), Foxp3-FITC (clone PCH101), IL-22-APC, IL-6 PerCP (both from eBioscience), CD68-APC (Biolegend, San Diego, CA). Mouse LPMCs and TILs were stained with the following monoclonal antibodies: CD45-APC-Cy7, CD4-PerCP, CD8α-FITC, CD49b-PE (clone DX5), FoxP3-APC, IL-17A-PE, IFN-γ-PE-Cy7 (both from BD Pharmingen), perforin-APC, granzyme B-PE-Cy7 (all from eBioscience). In parallel, cells were stained with the respective control isotype antibodies.
9
Multiparametric Immune Cell Profiling
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To examine the expression of surface markers and intracellular molecules, cells were incubated with FcR blocking reagent (Miltenyi, Germany) for 10 min followed by primary antibodies on ice in the dark for 30 min. The antibodies used for surface marker analysis included anti-human CD4-FITC, CXCR5-PE, PD-1-PE-cy7, BCL-6-APC, CD25-PE, CD19-FITC, CD138-PE (BD Pharmingen, USA), and anti-mouse CD4-FITC, CXCR5-APC, PD-1-PE, and CD138-PE (eBioscience, USA). For intracellular staining, cells were cytofixed and cytopermed using the CytofixCytoperm Plus kit (BD Pharmingen, USA) and stained with intracellular antibodies, including anti-human IL-17-APC, IFN-γ-PE, IL-4-PE-cy5, Foxp3-APC (BD Pharmingen, USA), 5-mC and 5-hmC (Abcam, USA) for an additional 30 min on ice in the dark. For some experiments, apoptosis was detected using a FITC Annexin V Apoptosis Detection Kit II (BD Pharmingen, USA). Data were acquired by flow cytometry (BD, Canto II, USA) and analyzed using FlowJo (Tree Star, USA).
10
Flow Cytometry Analysis of Splenic DC Subsets
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Anti-mouse CD11c-FITC, CD8-PE-Cy7, TCRβ-PE-Cy5.5, CD11b-Pacific Blue, B220-PE-Cy7, CD4-Pacific Blue, Foxp3-APC and CD86-PE antibodies were purchased from BD Biosciences. MHCII-AF700 and CD11c-Pacific Blue were purchased from Biolegend. 2×106 cells were stained in PBS containing 5% fetal calf serum (FCS) and 0.1% w/v sodium azide at 4°C for 30 min, washed with the same buffer and fixed with 1% paraformaldehyde containing 2 mM EDTA. Data were acquired on 4-laser LSR II using FACSDiva software (Becton Dickinson) and analyzed using Flow Jo 9.0 software (Tree Star, OR). For splenic DC subset analyses, TCRβ- CD11c+ cells were gated on and the MHCII and CD86 expression levels evaluated in CD11c+CD8+ and CD11c+CD11b+ cells.
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