Inflammatory cells in lung tissue were analyzed to identify eosinophils (CD45+CD11b+Ly6C-SiglecF+), Th17 cells (CD45+CD4+IL-17A+), regulatory T (Treg) cells (CD45+CD4+CD25+Foxp3+) by flow cytometry as previously described (Zhao et al., 2013 (link); Cossarizza et al., 2017 (link); Dong et al., 2018 (link)). Briefly, the excised right lung was digested by using lung dissociation kits (MiltenyiBiotec Technology & Trading Co. Ltd, Shanghai, China) to prepare single cell suspensions following the instructions. And then the cells were stained with the monoclonal anti-murine fluorochrome-conjugated Abs and detected by an Attune NxT instrument (Life Technology). Besides, the antibodies used were bought from BioLegend, including eflour506-labeled anti-CD45, Zombie NIR™ Fixable Viability Kit, Brilliant Violet 421™-labeled anti-CD11b, APC-labeled anti-Siglec-F, PE-labeled anti-Ly-6C, APC-labeled anti-CD25, FITC-labeled anti-CD4, PE-labeled anti-IL-17. PE-labeled anti-Foxp3 was purchased from eBioscience.
Pe labeled anti foxp3
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PE-labeled anti-FoxP3 is a flow cytometry reagent used for the detection and analysis of FoxP3-expressing cells. FoxP3 is a transcription factor that is a key marker for regulatory T cells (Tregs). The PE-labeled anti-FoxP3 antibody can be used to identify and quantify Tregs in various sample types, such as peripheral blood, lymphoid tissues, or cell cultures.
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Lab products found in correlation
Fitc labeled anti cd4, by Thermo Fisher Scientific (3 mentions)
Fixation permeabilization kit, by BD (2 mentions)
Pe cy7 labeled anti cd3, by Thermo Fisher Scientific (2 mentions)
Pe cy5 labeled anti cd8, by Thermo Fisher Scientific (2 mentions)
Cytexpert software, by Beckman Coulter (2 mentions)
Zombie nir fixable viability kit, by BioLegend (1 mentions)
Attune nxt instrument, by Thermo Fisher Scientific (1 mentions)
Brilliant violet 421 labeled anti cd11b, by BioLegend (1 mentions)
Fitc labeled anti cd4, by BioLegend (1 mentions)
Intracellular fixation buffer, by Thermo Fisher Scientific (1 mentions)
Foxp3 transcription factor staining buffer, by Thermo Fisher Scientific (1 mentions)
Apc labeled anti cd3, by BD (1 mentions)
Fitc labeled anti cd11c, by BD (1 mentions)
Fitc labeled anti cd8, by BD (1 mentions)
Pe labeled anti cd4, by BD (1 mentions)
Fitc labeled anti igg1, by BD (1 mentions)
Pe labeled anti cd11b, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
6 protocols using pe labeled anti foxp3
1
Characterizing Immune Cell Populations in Lung Tissue
2
Quantification of Murine CD4+ Treg Cells
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The isolated liver CD4+ T cells from individual mice were stained with FITC-labeled anti-CD4 (clone GK1.5, Ebioscience, USA) or APC-labeled anti-CD25 (clone PC61.5, Ebioscience, USA) for 30 minutes in the dark. After being washed, the cells were fixed with Intracellular Fixation buffer (Ebioscience, USA) and permeabilized with Foxp3/Transcription Factor Staining Buffer (Ebioscience, USA), followed by intracellularly staining with PE-labeled anti-FOXP3 (Ebioscience, USA). After being washed, the percentages of CD4+CD25+FOXP3+ Tregs in total CD4+ T cells were analyzed by flow cytometry in a flow cytometry machine (ACEA NovoCyte, USA or Beckman, USA). The data were analyzed by NovoExpressTM software or FlowJo software.
3
Immune Cell Profiling in MRL.Fas(lpr) Mice
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Spleens were isolated from MRL.Faslpr mice at 25 weeks of age, and immune cell composition was analyzed by flow cytometry. Spleen cells were stained with the following monoclonal antibodies in 0.5% BSA/PBS at 4°C for 15 min: FITC-labeled anti-B220, APC-labeled anti-CD3, PE-labeled anti-CD4, FITC-labeled anti-CD8, FITC-labeled anti-CD11c, PE-labeled anti-CD11b, APC-labeled anti-CD138, FITC-labeled anti-IgG1 (BD Biosciences, San Jose, CA, USA), FITC-labeled anti-CD4, and PE-labeled anti-Foxp3 (eBioscience). Data were collected using FACSCalibur and analyzed with CellQuest Pro software (BD Biosciences).
4
Identification of Regulatory T Cells
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Cells in the basal compartments in all
coculture setups were stained with surface markers FITC-labeled anti-CD4
(Cell Signaling Technology), APC-labeled anti-CD25 (Cell Signaling
Technology), PE-Cy7-labeled anti-CD127 (eBioScience), and Treg-specific
transcription factor PE-labeled anti-FoxP3 (eBioscience) antibodies
for identification of Treg cells. The eBioscience Foxp3/Transcription
Factor Staining Buffer Set was purchased from ThermoFisher. Single
cell suspensions were resuspended at 1 × 106 cells/mL
and were initially stained with the surface markers for at least 30
min in 4 °C. Cells were then fixed and permeabilized. Intracellular
staining of Foxp3 was then carried out according to the manufacturer’s
protocol. Flow cytometry was performed with BD LSR Fortessa, and the
results were analyzed using the Flowjo software.
coculture setups were stained with surface markers FITC-labeled anti-CD4
(Cell Signaling Technology), APC-labeled anti-CD25 (Cell Signaling
Technology), PE-Cy7-labeled anti-CD127 (eBioScience), and Treg-specific
transcription factor PE-labeled anti-FoxP3 (eBioscience) antibodies
for identification of Treg cells. The eBioscience Foxp3/Transcription
Factor Staining Buffer Set was purchased from ThermoFisher. Single
cell suspensions were resuspended at 1 × 106 cells/mL
and were initially stained with the surface markers for at least 30
min in 4 °C. Cells were then fixed and permeabilized. Intracellular
staining of Foxp3 was then carried out according to the manufacturer’s
protocol. Flow cytometry was performed with BD LSR Fortessa, and the
results were analyzed using the Flowjo software.
5
Quantification of Tumor-Infiltrating Lymphocytes
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Tumors isolated from the mice were digested mechanically to obtain single-cell suspensions. The cell suspensions were surface stained with PE-Cy7-labeled anti-CD3 (eBioscience, San Diego, CA, USA), FITC-labeled anti-CD4 (eBioscience, San Diego, CA, USA) and PE-Cy5-labeled anti-CD8 (eBioscience, San Diego, CA, USA) monoclonal antibodies, and then treated with Fixation/Permeabilization Kit (BD Biosciences, Franklin Lakes, NJ, USA). Then, the cells were stained intracellularly with PE-labeled anti-FoxP3 (eBioscience, San Diego, CA, USA) and Pacific Blue-labeled anti-Granzyme B monoclonal antibodies (Biolegend, San Diego, CA, USA). The stained cells were analyzed with CytoFLEX LX flow cytometer (Beckman Coulter, Brea, CA, USA). Data were analyzed using CytExpert software (Beckman Coulter, Brea, CA, USA).
6
Tumor-Infiltrating Immune Cell Profiling
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Tumors isolated from the mice were digested mechanically to obtain single-cell suspensions. The cell suspensions were surface stained with PE-Cy7-labeled anti-CD3 (eBioscience, San Diego, CA, USA), FITClabeled anti-CD4 (eBioscience, San Diego, CA, USA) and PE-Cy5-labeled anti-CD8 (eBioscience, San Diego, CA, USA) monoclonal antibodies, and then treated with Fixation/Permeabilization Kit (BD Biosciences, Franklin Lakes, NJ, USA). Then, the cells were stained intracellularly with PE-labeled anti-FoxP3 (eBioscience, San Diego, CA, USA) and Paci c Blue-labeled anti-Granzyme B monoclonal antibodies (Biolegend, San Diego, CA, USA). The stained cells were analyzed with CytoFLEX LX ow cytometer (Beckman Coulter, Brea, CA, USA). Data were analyzed using CytExpert software (Beckman Coulter, Brea, CA, USA).
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