After being harvested and weighed, tumor tissues were cut into small pieces and mixed with IV collagenase for 2 h. Tissues were homogenized and filtered through a 70 μm filter to remove remaining undigested tissue. The single cell suspension was centrifuged at 500 g for 5 min, incubated with 2 μl extracellular antibody for 30 min, and then fixed with 4% paraformaldehyde for 20 min. Finally, the suspension was incubated with intracellular antibody for another 30 min before being tested by flow cytometry (NovoCyte, ACEA Biosciences, San Diego, USA). APC-Anti-CD4 (#553051), PE-Cy7-anti-CD25(#552880), PE-anti-FOXP3(#563101), PE-Cy7-anti-CD11b (#552850) and FITC-anti-Gr-1(#553126) were purchased from BD pharmingen™.
Anti foxp3 pe
Manufactured by BD
Sourced in United States
Anti-Foxp3-PE is a laboratory reagent used for the detection and analysis of Foxp3-expressing cells, which are a key marker of regulatory T cells (Tregs). It utilizes a phycoerythrin (PE) fluorescent label to enable flow cytometric identification and quantification of Foxp3-positive cell populations.
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Lab products found in correlation
Anti cd4 fitc, by BD (21 mentions)
Anti cd25 apc, by BD (15 mentions)
Anti cd25 pe cy7, by BD (8 mentions)
Anti il 17a pe, by BD (7 mentions)
Anti cd3 fitc, by BD (6 mentions)
Anti cd4 percp, by BD (5 mentions)
Anti cd4 apc, by BD (4 mentions)
Anti cd8 apc, by BD (4 mentions)
Facscalibur, by BD (4 mentions)
Anti ifn γ pe, by BD (4 mentions)
Anti cd19 apc, by BD (3 mentions)
Anti cd8 pe, by BD (3 mentions)
Facscalibur cytometer, by BD (3 mentions)
Anti cd11c fitc, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Ionomycin, by Merck Group (3 mentions)
Anti cd11b pe cy7, by BD (2 mentions)
Sodium bicarbonate, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Iscove s modified dulbecco s medium, by Thermo Fisher Scientific (2 mentions)
51 protocols using anti foxp3 pe
1
Flow Cytometry Analysis of Tumor Immune Cells
2
Cell Line Maintenance and Characterization
3
Cell Line Maintenance and Characterization
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Human cervical carcinoma cell line (HeLa), hepatocellular carcinoma cell line (Huh7), and AAV packaging cell line (AAV293) was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were maintained as monolayer cultures in Iscove’s modified Dulbecco’s medium (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco), 10 μg/mL piperacillin (MP Biomedicals, Illkirch, France), 10 μg/mL ciprofloxacin (HiMedia Laboratories, Mumbai, India) and sodium bicarbonate (Sigma-Aldrich, St Louis, MO, USA). The cell lines used in this study were authenticated by short tandem repeat profiling (data not shown). Antibodies such as FITC-anti CD3, PerCP-anti CD4, PE-anti CD8, APC-anti CD19, PE-anti Foxp3, and APC-anti CD25 for immune assays were from BD Biosciences (Franklin Lakes, NJ, USA).
4
Comprehensive Immune Cell Profiling and Thyroid Hormone Receptor Analysis
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PE anti-CD16 (catalog 555407), PerCP anti-CD14 (catalog 340585), and APC anti-CD56 (catalog 555518) were used to identify monocytes and NK cells. FITC anti-CCR7 (catalog 561271), PE anti-CD27 (catalog 555441), PerCP anti-CD3 (catalog 555332), and APC anti-CD56 (catalog 555518) were used to identify the T cell and NK cell populations. FITC anti-CD4 (catalog 555346), PE anti-Foxp3 (catalog 560046), and APC anti-CD25 (catalog 555434) were used to label Tregs (all antibodies used were obtained from BD Biosciences). To determine the expression of thyroid hormone receptors, we used Alexa Fluor 488 anti–thyroid hormone receptor β and FITC-thyroid hormone receptor alpha (Bioss Antibodies) and performed intracellular staining using an intracellular staining kit (BioLegend). In addition, we used PE anti–human GPR83 (BioLegend) to determine the expression of glucocorticoid receptors.
5
Multiparameter Immune Cell Analysis
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FITC anti-CD4, PE anti-FoxP3 and PE and APC anti- ICOS (all BD Biosciences) and IL-23R (R and D systems) were used. To determine the absolute CD4 and CD8 counts BD Trucount tubes and BD Multitest antibodies were used. For neutralization experiments, we used anti- PD1, and isotype control antibodies (Bio legend).
6
Fisetin-Mediated Immunomodulation Assay
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Fisetin (>98% pure, catalog number: 528483, Figure 1 ) was obtained from Sigma-Aldrich and diluted in culture media containing 0.1% DMSO, sterile filtered, and stored at 4°C prior to utilization. An equivalent DMSO concentration was used for all control animals. Cyclophosphamide (CTX) and 2,4-dinitrofluorobenzene (DNFB) were procured from Sigma (MO, USA). RPMI-1640 was from HyClone (Logan, UT). Moreover, BioLegend (CA, USA) supplied IL-6, IL-17, TGF-β, TNF-α, and IL-10 ELISA kits. PE-anti-IL-17A, FITC-anti-CD4, APC-anti-CD25, and PE-anti-Foxp3 were from BD Pharmingen.
7
Multi-Dimensional Immune Cell Profiling
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Single-cell suspensions were stained for 30 min using the following fluorophore-conjugated antibodies: FITC anti-CD3 (BD Biosciences Cat# 555332, RRID:AB_395739), APC/Cy7 anti-CD4 (BD Biosciences Cat# 341095, RRID:AB_400218), Percp-Cy5.5 anti-CXCR5 (BD Biosciences Cat# 562781, RRID:AB_2313576), PE anti-PD-1 (BD Biosciences Cat# 560795, RRID:AB_2033989), PE/Cy7 anti-CCR6 (BD Biosciences Cat# 560620, RRID:AB_1727440), APC anti-CCR6 (BD Biosciences Cat# 560619, RRID:AB_1727439), PECy7 anti-CXCR3 (BD Biosciences Cat# 560831, RRID:AB_2033944), APC anti-CXCR3 (BD Biosciences Cat# 565223, RRID:AB_2687488), FITC anti-CD19 (BD Biosciences Cat# 555412, RRID:AB_395812), BV421 anti-CD27 (BioLegend Cat# 302823, RRID:AB_10900425), APC anti-CD38 (BD Biosciences Cat# 555462, RRID:AB_398599), PECy7 anti-IgD (BD Biosciences Cat# 555776, RRID:AB_396111), and PE anti-CD138 (BD Biosciences Cat# 552026, RRID:AB_394323). For intracellular staining, cells were permeabilized using the Foxp3 Staining Buffer Set (eBioscience), then stained with PECy7 anti-Ki67 (BD Biosciences Cat# 561283, Clone B56), PE anti-Foxp3 (BD Biosciences Cat# 560082, RRID:AB_1645509), and PE anti-Bcl2 (BD Biosciences Cat# 340576, RRID:AB_400061). All samples were analyzed with an LSRFortessa flow cytometer (Beckton Dickinson), and data were processed with FlowJo software, version 10 (TreeStar, Inc.).
8
Comprehensive Flow Cytometry Immunophenotyping
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Multi-parameter cytofluorometric analysis was performed to phenotype PBLs. The following conjugated monoclonal antibodies were purchased from BD Biosciences: peridinin chlorophyll protein complex–cyanine (PerCP-5–5)-anti-CD3, fluorescein isothiocyanate (FITC)-anti-CD4, allophycocyanin–cyanine (APC-Cy7)-anti-CD8, phycoerythrin–cyanine (PE-Cy7)-anti-CD25, PE–anti-FOXP3, PE-anti-CD45RO, FITC-anti-CD45RA, allophycocyanin (APC)-anti-CD45RA, and FITC-anti-Ki67. Cryopreserved cells were resuspended in a staining buffer supplied by the manufacturer (BD Biosciences, San Jose, CA) and stained for 20 min at 4 °C. The expression of FOXP3 was detected by intracellular staining of cells pre-stained with other markers and cell permeabilization was performed according to the manufacturer’s instructions (BD Biosciences). Samples were run on a FACSCanto flow cytometer (Becton Dickinson) with at least 5 × 104 events acquired per parameter. Sequential gating was used to discriminate lymphocyte subsets, as previously described.42 (link) Flow cytometry data were analyzed using the FloJo software package (Treestar).
9
Characterization of T-cell Subsets in Autoimmune Hepatitis
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Peripheral blood mononuclear cells (PBMCs) and spleen mononuclear cell (SMNCs) were isolated from AIH patients and EAH mice by density gradient centrifugation according to the manufacturer's instruction. To detect different T‐cell subsets, PBMCs or SMNCs (1 × 106/tube) were stained with BV510‐anti‐CD4 (0.2 mg/mL), PerCP‐Cy5.5‐anti‐CXCR5 (0.1 mg/mL), PE‐Cy7‐anti‐CD25 (0.05 mg/mL), BV421‐anti‐ICOS (0.05 mg/mL), FITC‐anti‐PD‐1 (0.05 mg/mL), APC‐anti‐CTLA‐4(0.2 mg/mL) (BD Biosciences) in the dark at 4°C for 30 minutes, fixed, permeabilized using transcription factor Fix/Perm buffer, and stained with antibodies for intracellular proteins including PE‐anti‐FoxP3(0.1 mg/mL), PE‐CF594‐anti‐IL‐10(0.05 mg/mL) and Alexa Fluor 647‐anti‐IL‐21(0.1 mg/mL) (BD Biosciences). After washing with transcription factor Perm/wash buffer (BD Biosciences), the numbers of each phenotype of TFR and TFH cells in AIH patients and the frequency of GITR+TFR and GITR‐TFH in EAH mice were characterized by flow cytometry analysis.
10
Phenotypic Analysis of T cells
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