105 PBMCs were resuspended in 100uL of PBS for labeling with human cell-surface antibodies (all from eBioscience) in two different conditions: (1) antiCD25 PECy7 (BC96), antiCD127 PerCPCy5.5 (eBioRDR5), antiCD4 FITC (RPA-T4;) together, or (2) antiCD4 FITC (RPA-T4) and antiCD45RA PerCPCy5.5 (HI100). Cells were then permeabilized with the “Human FoxP3 Buffer Set” BD-Pharmingen (San Diego, CA) according to the manufacturers' recommendation and labeled with FOXP3 PE (236A). A hundred thousand events per sample were acquired by Attune® (Thermo Fisher Scientific) flow cytometer. Analysis was done with FlowJo 7.6.5 (Tree Star® Inc.) (Figure 2 ). Previous antibody titrations and FMO (Fluorescence Minus One) control were also performed, ideal for showing gating boundaries in multicolor flow cytometry [27 (link)](Figure S1 ).
Human foxp3 buffer set
Manufactured by BD
Sourced in United States
The BD Human FoxP3 Buffer Set is a collection of reagents designed for the detection and analysis of human FoxP3-expressing cells. The set includes a fixation/permeabilization concentrate, a fixation/permeabilization diluent, and a permeabilization buffer. These components are essential for the intracellular staining and flow cytometric analysis of FoxP3, a transcription factor that is a key marker for regulatory T cells.
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Lab products found in correlation
Facsdiva software, by BD (4 mentions)
Cytofix cytoperm, by BD (3 mentions)
Facscanto 2, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
Fitc anti human flow kit, by BioLegend (2 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cyan adp flow cytometer, by Beckman Coulter (2 mentions)
Facsverse flow cytometer, by BD (2 mentions)
Cd25 pe, by BD (2 mentions)
Facsuite software, by BD (2 mentions)
Cd4 percp cy5, by BD (2 mentions)
Facsaria, by BD (2 mentions)
Foxp3 pe, by BD (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Lsrfortessa, by BD (1 mentions)
Rpa t8, by BD (1 mentions)
37 protocols using human foxp3 buffer set
1
Multicolor Flow Cytometry Analysis of T-cell Subsets
2
Flow Cytometry Analysis of RRMS Patient Sera on Brain Endothelial Cells
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hCMEC/D3 cells incubated (16 h) in EBM2 medium containing 20% sera from RRMS patients or matched HD were collected in FACS buffer, fixed with 2% formaldehyde (Sigma-Aldrich) for 10 min at RT and washed with FACS buffer (1% BSA in PBS). ICAM-1 APC (1:100 Bioscience), VCAM-1 Alexa fluor 488 (1:100, eBioscience), Occludin AF 405 (1:100 eBioscience) VeCadherin Alexa fluor 488 (1:100, eBioscience) and P-glycoprotein Alexa fluor 488 (1:100 ebioscience) were added for 30 min at room temperature. Between each step, samples were vortexed and centrifuged for 5 min at 4 °C. Finally, cells were suspended in PBS and analysed by FACS using a LSR-Fortessa (BD Biosciences), with 10,000 events collected on gated cell population.
The expression of markers in PBMCs were measured on fixed cells incubated with staining buffer (PBS + 2% BSA). Cell surface staining for CD4-AF648 was carried out at room temperature for 30 min. After washing, the cells were permeabilised using the human Foxp3 buffer set (BD Biosciences) for 20 min followed by a wash step and staining within intracellular markers Foxp3-PE and RoRγt-BV421. After a final wash, cells were suspended in PBS for FACS analysis. All data was analysed using FlowJo software version 10.
The expression of markers in PBMCs were measured on fixed cells incubated with staining buffer (PBS + 2% BSA). Cell surface staining for CD4-AF648 was carried out at room temperature for 30 min. After washing, the cells were permeabilised using the human Foxp3 buffer set (BD Biosciences) for 20 min followed by a wash step and staining within intracellular markers Foxp3-PE and RoRγt-BV421. After a final wash, cells were suspended in PBS for FACS analysis. All data was analysed using FlowJo software version 10.
3
Flow Cytometry Immunophenotyping Protocol
4
Isolation and Characterization of Ascites Cells
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Cells from the patient ascites fluid were isolated as described previously [31 (link)]. In brief: after filtration of the ascites fluid through a 40 μM mesh, cells were sedimented by 10 min centrifugation at 350 g. Then, cells were fixed and permeabilised using the Human FoxP3 Buffer Set (BD), stained with CD3-Pacific Blue (UCHT1, BD Biosciences, AB_397038), CD4-PerCP (L200, BD Biosciences, AB_393791), CD8-PE-Cy7 (RPA-T8, BD Biosciences, AB_396856) and CD45RA-APC-H7 (HI100, BD Biosciences, AB_1727497) and run on a FACSCanto II (BD Biosciences). Data visualization and analysis was done in FlowJo (V10) (FlowJo, LLC).
5
Flow Cytometric Analysis of Immune Cells
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The following monoclonal antibodies were used for flow cytometric analysis of immune cells: anti-mouse CD4 (BD Pharmingen, Cat. 553088), anti-mouse IFN-γ (BD Pharmingen, Cat. 560660), anti-mouse IL-4 (BD Pharmingen, Cat. 560699), IL-17a (BD Pharmingen, Cat. 561020), CD8 (Biolegend, Cat. 100744), B220 (BD Pharmingen, Cat. 563894), CD11c (BD Pharmingen, Cat. 746392), CXCR5 (Biolegend, Cat. 560617), CD138 (BD Pharmingen, Cat. 568626), IgD (BD Pharmingen, Cat. 553510), CD19 (Biolegend, Cat. 115530), CD45 (BD Pharmingen, Cat. 560510), CD11b (BD Pharmingen, Cat. 564454), and Gr-1 (Biolegend, Cat. 108410). For cytokine analysis, cells were stimulated in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, HyClone), PMA, ionomycin, and GolgiPlug (BD Biosciences, Cat. 550583) at 37°C and 5% CO2 for 6 h. For intracellular staining, cells were fixed and permeabilized with a Human Foxp3 Buffer Set (BD Biosciences, Cat. 562574) or Cytofix/Cytoperm (BD Biosciences, 554722) according to the manufacturer’s instructions.
6
Analysis of T Cell Subsets and Activation Markers
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Recombinant human TNF was purchased from Leinco Technologies (St Louis, MO, USA), and recombinant human IL-2 was purchased from Sigma-Aldrich (St Louis, MO, USA). Monoclonal antibodies against TNFR2 were produced in house or purchased from commercial vendors as previously described.8 (link) Fluorochrome-conjugated mAbs against human CD4 (RPA-T4), CD25 (M-A251), CD45RA (HI100, 2H2), CD45RO (UCHL1, 4HB), CD127 (hIL-7R-M21), HLA-DR (L243 (G46-6)) were purchased from BD Biosciences. Fluorochrome-conjugated monoclonal antibodies against CD4 (S3.5, Invitrogen, Carlsbad, CA, USA), CD120a (16803 R&D systems), CD120b (22235, R&D systems, Minneapolis, MN, USA), FOXP3 (259D, Biolegend, San Diego, CA, USA) were also used in this study.
Intracellular staining of FOXP3 and CD152 was performed using either FOXP3 Fix/Perm Buffer set (Biolegend) or Human FoxP3 Buffer set (BD Biosciences) according to the manufacturer's instructions. Flow cytometric data were obtained using FACSCalibur (BD Biosciences) flow cytometer. All the data were analyzed with Cellquest Software (BD Biosciences, San Jose, CA, USA).
Intracellular staining of FOXP3 and CD152 was performed using either FOXP3 Fix/Perm Buffer set (Biolegend) or Human FoxP3 Buffer set (BD Biosciences) according to the manufacturer's instructions. Flow cytometric data were obtained using FACSCalibur (BD Biosciences) flow cytometer. All the data were analyzed with Cellquest Software (BD Biosciences, San Jose, CA, USA).
7
Regulatory T Cell Phenotyping and Cytokine Analysis
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To detect Tregs, intracellular staining of CD4+ CD25+ T cells was performed using a human FOXP3 buffer set (BD Biosciences) and anti-human FOXP3-PE antibody (BD Pharmingen) according to the manufacturer’s instructions. For intracellular cytokine analysis, CD4 T cells cultured alone or cocultured with ASCs were restimulated with leukocyte activation cocktail (BD Biosciences) for 2-3 h on the day of analysis. The T cells were then harvested, fixed, and permeabilized using a BD Cytofix/Cytoperm kit (BD Biosciences). The T cells were stained with BV605-conjugated anti-human IFN-gamma or APC-conjugated anti-human IL-10 (BD Biosciences) and analyzed using a CytoFlex S flow cytometer.
8
Treg Cell Enumeration by Flow Cytometry
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Antibodies used for Treg cell assessment in flow cytometry were purchased from BD Pharmingen (Becton Dickinson, San Jose, CA, USA, BD); anti-CD4 conjugated to PE-Cy5, anti-CD25 (alpha chain of IL-2 receptor) conjugated to allophycocyanin (APC) and anti-FoxP3 conjugated to phycoerythrin (PE). The staining process was as follows: small aliquots (100 μl) of whole blood containing K3EDTA were incubated for 30 min with anti-CD4 and anti-CD25, and red blood cells were lysed subsequently. After surface staining, cells were fixed and permeabilized using the Fixation/Permeabilization Kit (BD, Human FoxP3 Buffer Set) for intracellular staining. Briefly, cells were fixed in FoxP3 Buffer A, for 15 minutes at room temperature (RT) and the cells were washed. To permeabilize cells they were incubated for 30 minutes in human FoxP3 Buffer B. Finally, after washing, cells were stained with 10 μl of PE Mouse anti-Human FoxP3. All of the process was protected from light.
Firstly gating was done on CD4+ cells then detection of CD25high, FoxP3+ within the population followed. Isotype controls were used to discriminate unstained cells. Analysis was performed using a flow cytometer (PARTEC, Münster, Germany). The results were analyzed using Flow Jo software version 7.6.1 (Tree Star, Ashland, OR, USA).
Firstly gating was done on CD4+ cells then detection of CD25high, FoxP3+ within the population followed. Isotype controls were used to discriminate unstained cells. Analysis was performed using a flow cytometer (PARTEC, Münster, Germany). The results were analyzed using Flow Jo software version 7.6.1 (Tree Star, Ashland, OR, USA).
9
Analysis of CD4+ and Treg Cells
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Treated PBMCs were collected and washed twice with PBS and labelled with anti‐CD4‐FITC, anti‐CD25‐APC and anti‐Foxp3‐PE antibodies (BD Bioscience, San Diego, CA, USA) following the manufacturer's instructions of the human Foxp3 Buffer Set (BD Bioscience). The CD4+ cell population was calculated as the percentage of cells positive for CD4 within the total lymphocyte population. The Treg cell population was calculated as the percentage of cells positive for CD4, CD25, and Foxp3 staining (CD4+ CD25+ Foxp3+) among the total lymphocyte population. Flow cytometry analysis was performed with FACSCalibur cytometer and CellQuest Pro (BD Biosciences) software.
10
Isolation and Characterization of Human Regulatory T Cells
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Tregs were isolated with the CD4+CD25+highCD127−/dim Regulatory T Cell Isolation Kit II (human) (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's instructions. In brief, peripheral blood mononuclear cells (PBMCs) were isolated with a Ficoll gradient (Biochrom AG, Berlin, Germany). The CD4+CD25+CD127− T Cell Biotin-Antibody Cocktail II was used to negatively enrich for CD4+CD127− cells followed by a positive selection of CD25+ cells.
To assess the purity of these CD4+CD25+highCD127−/dim cells, samples and PBMCs were tested by flow cytometry. Hence, cells were incubated with the appropriate amount of extracellular antibody and prepared for intracellular staining with FoxP3-Alexa Fluor 647 (BioLegend) using the Human FoxP3 Buffer Set (BD Biosciences). For extracellular staining, CD25-PE-Cy7, CD4-FITC (both BD Biosciences), and CD127-Pacific Blue (BioLegend) were used. In all experiments, the purity of CD4+CD25+highCD127−/dimFoxP3+ cells was ≥95%.
To assess the purity of these CD4+CD25+highCD127−/dim cells, samples and PBMCs were tested by flow cytometry. Hence, cells were incubated with the appropriate amount of extracellular antibody and prepared for intracellular staining with FoxP3-Alexa Fluor 647 (BioLegend) using the Human FoxP3 Buffer Set (BD Biosciences). For extracellular staining, CD25-PE-Cy7, CD4-FITC (both BD Biosciences), and CD127-Pacific Blue (BioLegend) were used. In all experiments, the purity of CD4+CD25+highCD127−/dimFoxP3+ cells was ≥95%.
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