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Flow cytometry of GATA transcription factors
Author(s) -
Miura Toshihiko,
Yokoyama Hisayuki,
Minegishi Naoko,
Sasaki Takeshi,
Kaku Mitsuo,
Harigae Hideo
Publication year - 2003
Publication title -
cytometry part b: clinical cytometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.646
H-Index - 61
eISSN - 1552-4957
pISSN - 1552-4949
DOI - 10.1002/cyto.b.10047
Subject(s) - flow cytometry , western blot , immunofluorescence , gata transcription factor , population , transfection , gata2 , biology , microbiology and biotechnology , cell , cell culture , antibody , haematopoiesis , gene expression , immunology , gene , stem cell , genetics , medicine , promoter , environmental health
Background Although GATA‐1 and GATA‐2 have been shown to play an important role in hematopoiesis, the expression levels of these GATA proteins in the targeted cell population of clinical samples have not been studied. We applied flow cytometry (FCM) to examine the expression levels of these GATA proteins in the selected subpopulation in heterogeneous blood cells. Methods Cells were treated with a fixing solution and methanol followed by staining with specific antibodies to GATA proteins in a permeabilizing solution. Immunofluorescence microscopy and Northern blot analysis using GATA‐1 and GATA‐2 transfected cell lines and various leukemic cell lines were used to confirm the specificity of this method. Subsequently, the method was applied in two‐parameter studies combining GATA expression with surface marker expression in clinical samples. Results The positive signals were specifically detected in transfected cells and leukemic cell lines by FCM in agreement with the results of Northern blot and immunofluorescence microscopy. The expression of these GATA factors in the targeted cell population was easily detectable by gating with lineage‐specific cell surface markers. When the expression of these GATA proteins was examined in glycophorin A‐positive cells in clinical samples, the level of GATA‐1 was markedly different among the samples. Conclusions This detection system is useful to evaluate the relative expression level of each GATA protein in the targeted cell population among heterogeneous cells, and the results suggest an aberrant expression of GATA factors in hematological diseases. Cytometry Part B (Clin. Cytometry) 56B:1–7, 2003. © 2003 Wiley‐Liss, Inc.