Detection of intracellular antigens by flow cytometry: comparison of two chemical methods and microwave heating.
Detection of intracellular antigens by flow cytometry requires effective fixation and permeabilization of the cell membrane. This study compares three fixation/permeabilization techniques: two commercial chemical reagents, the ORTHOPermeaFix (OPF) and the FIX&PERM Cell Permeabilization Kit (F&P), and a novel method based on microwave heating (MWH). They have been applied to the detection of two nuclear (p53 and rb/p105) and two cytoplasmic (bcl-2 and mdr-1/gp-170) antigens, using positive- and negative-control cell lines and peripheral blood mononuclear cells. Western blotting was performed as a control of protein expression. For the four antigens assessed, cellular morphology, discrimination between intact cells and debris, percentage of positive cells, and mean fluorescence intensity were examined. For this last parameter, the assessment of the MWH technique was performed using SD and a graphical approach inspired by the concepts described by Bland and Altman (Lancet 1986;346: 1085-7) as well as Petersen et al. (Clin Chem 1997;43: 2039-46). The statistical analysis shows that MWH is comparable to the commercial methods and that its reproducibility is also equivalent to OPF and F&P. As assessed for some of the most clinically relevant intracytoplasmic and intranuclear antigens, the MWH method appears to be a valuable and inexpensive alternative. It is worth noting that, unlike commercial reagents, MWH altered surface antigens. Interestingly, this feature, which would prevent cell selection on the basis of combined membrane and intracellular epitopes, is associated with a decrease of nonspecific background fluorescence.
Millard
I
I
Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Clinical Biochemistry Department, Clos-Chapelle-aux-Champs, Brussels, Belgium.
Degrave
E
E
Philippe
M
M
Gala
J L
JL
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000528:radiation effects; D000941:Antigens / Q000032:analysis* / Q000378:metabolism / Q000528:radiation effects; D002460:Cell Line; D003593:Cytoplasm / Q000276:immunology / Q000378:metabolism / Q000528:radiation effects; D000939:Epitopes / Q000032:analysis / Q000378:metabolism / Q000528:radiation effects; D005434:Flow Cytometry / Q000379:methods; D019450:Genes, MDR / Q000276:immunology / Q000528:radiation effects; D006801:Humans; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism / Q000528:radiation effects / Q000648:ultrastructure; D008872:Microwaves; D009687:Nuclear Proteins / Q000032:analysis / Q000276:immunology / Q000528:radiation effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis* / Q000276:immunology / Q000528:radiation effects; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D016160:Retinoblastoma Protein / Q000032:analysis* / Q000276:immunology / Q000528:radiation effects; D016159:Tumor Suppressor Protein p53 / Q000032:analysis* / Q000276:immunology / Q000528:radiation effects
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000941:Antigens; D000939:Epitopes; D009687:Nuclear Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011933:Reagent Kits, Diagnostic; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53