title: An improved intracellular staining protocol for efficient detection of nuclear proteins in YFP-expressing cells.
abstract: Intracellular staining is a widely used flow cytometry (FCM)-based technique to detect the expression of cytoslio nucleic antigens. However, intracellular staining of cells expressing cytosolic fluorescent protein (FP) markers was proven to be problematic as significant loss of the FP-signal was routinely observed. Using splenocytes harvested from mice constitutively expressing the enhanced yellow fluorescent proteins (YFP) as a model, we modified the widely used intracellular staining protocol and successfully achieved simultaneous detection of both the nuclear proteins and YFP in T-regulatory cells. The improved protocol can be used to perform antibody-based intracellular characterization of FP-labeled target cells, while maintaining their fluorescent reporter signals for easy tracing and identification.
authors.affiliation: Division of Immunogenetics, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Grupillo
authors.forename: Robert
authors.initials: R
authors.lastname: Lakomy
authors.forename: Xuehui
authors.initials: X
authors.lastname: Geng
authors.forename: Alexis
authors.initials: A
authors.lastname: Styche
authors.forename: William A
authors.initials: WA
authors.lastname: Rudert
authors.forename: Massimo
authors.initials: M
authors.lastname: Trucco
authors.forename: Yong
authors.initials: Y
authors.lastname: Fan
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis*; D002463:Cell Membrane Permeability; D003593:Cytoplasm / Q000737:chemistry*; D008164:Luminescent Proteins / Q000032:analysis*; D051379:Mice; D009687:Nuclear Proteins / Q000032:analysis*; D013154:Spleen / Q000166:cytology; D013194:Staining and Labeling / Q000379:methods*; D050378:T-Lymphocytes, Regulatory / Q000166:cytology*; D016707:Tissue Fixation / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; C054562:yellow fluorescent protein, Bacteria