title: Validated protocol for FoxP3 reveals increased expression in type 1 diabetes patients.
abstract: BACKGROUND
  FoxP3 has become a key identifier of regulatory T cells. Investigators have used a variety of antibodies and methods for detecting FoxP3 by flow cytometry. To standardize FoxP3 antibody staining for use in clinical trial samples, we tested various antibodies from different vendors, cell preparation protocols and fix/perm reagents, and cell isolation procedures. Using this optimized staining protocol, we evaluated clinical specimens from patients with multiple sclerosis (MS) or type 1 diabetes.
  
  
  METHODS
  FoxP3 antibodies from eBioscience (236A/E7 and PCH101) and BioLegend (206D) were evaluated along with their respective methods and fix/perm reagents for preparation and staining of FoxP3 for flow cytometry. Fresh washed blood and frozen or fresh PBMC were evaluated. Upon optimization of the protocol, clinical samples (frozen PBMC) from patients with MS or type 1 diabetes and healthy control donors were evaluated with the BioLegend antibody.
  
  
  RESULTS
  Clone 206D from BioLegend yielded optimal staining and the fix/perm reagents from both eBioscience and BioLegend were comparable. Data were also comparable between cells separated by Ficoll (fresh or frozen) and washed blood samples, allowing this protocol to be applicable to different types of samples. We validated this protocol using clinical samples and saw a significant increase in FoxP3 expression in the patients with type 1 diabetes but not in the MS.
  
  
  CONCLUSIONS
  The results from this study will allow the assessment of FoxP3 by flow cytometry on samples from clinical sites that are analyzed in real time on fresh blood or frozen PBMC.
authors.affiliation: University of California San Francisco, Bethesda, Maryland 20814, USA.
authors.forename: Jean
authors.initials: J
authors.lastname: Grant
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Bourcier
authors.forename: Stephen
authors.initials: S
authors.lastname: Wallace
authors.forename: Dalin
authors.initials: D
authors.lastname: Pan
authors.forename: Alexis
authors.initials: A
authors.lastname: Conway
authors.forename: Vicki
authors.initials: V
authors.lastname: Seyfert-Margolis
authors.forename: Paul K
authors.initials: PK
authors.lastname: Wallace
mesh_terms: D000906:Antibodies; D015415:Biomarkers / Q000032:analysis / Q000097:blood; D002999:Clone Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D015925:Cryopreservation / Q000379:methods; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000175:diagnosis / Q000276:immunology*; D005362:Ficoll; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D051858:Forkhead Transcription Factors / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D007108:Immune Tolerance / Q000276:immunology; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000379:methods; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D016707:Tissue Fixation / Q000379:methods; D015854:Up-Regulation / Q000276:immunology
chemical_list: D000906:Antibodies; D015415:Biomarkers; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D005362:Ficoll