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Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells
Published on: May 6, 2013
Validated protocol for FoxP3 reveals increased expression in type 1 diabetes patients.
Jean Grant1, Katarzyna Bourcier, Stephen Wallace
1University of California San Francisco, Bethesda, Maryland 20814, USA.
Cytometry. Part B, Clinical Cytometry
|August 12, 2008
Summary
Standardizing FoxP3 antibody staining for regulatory T cells is crucial for clinical trials. This study optimized flow cytometry methods, identifying optimal reagents and protocols for reliable FoxP3 detection in various sample types.
Area of Science:
- Immunology
- Cell Biology
- Clinical Trials
Background:
- Forkhead box P3 (FoxP3) is a key marker for regulatory T cells.
- Flow cytometry is widely used for FoxP3 detection, but standardization across different antibodies and protocols is needed for clinical applications.
- Previous methods lacked standardization, impacting the reliability of FoxP3 analysis in clinical trial samples.
Purpose of the Study:
- To standardize FoxP3 antibody staining for flow cytometry in clinical trial samples.
- To evaluate and compare different FoxP3 antibodies, cell preparation methods, and fix/perm reagents.
- To validate an optimized protocol using clinical specimens from patients with autoimmune diseases.
Main Methods:
- Evaluated FoxP3 antibodies from eBioscience (236A/E7, PCH101) and BioLegend (206D).
- Assessed various cell preparation protocols, fix/perm reagents, and cell isolation procedures (Ficoll separation, washed blood).
- Validated the optimized protocol on frozen peripheral blood mononuclear cells (PBMC) from patients with multiple sclerosis (MS), type 1 diabetes, and healthy controls.
Main Results:
- BioLegend's 206D clone provided optimal FoxP3 staining.
- Fix/perm reagents from both eBioscience and BioLegend showed comparable results.
- The optimized protocol demonstrated applicability to fresh washed blood, fresh PBMC, and frozen PBMC.
- A significant increase in FoxP3 expression was observed in type 1 diabetes patients, but not in MS patients, compared to controls.
Conclusions:
- The developed protocol enables reliable assessment of FoxP3 expression by flow cytometry.
- This standardization is applicable to clinical sites analyzing fresh blood or frozen PBMC in real-time.
- The findings support the use of standardized FoxP3 staining for evaluating immune responses in autoimmune diseases.
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