Flow cytometry of GATA transcription factors.
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.
Miura
Toshihiko
T
Department of Molecular Diagnostics, Tohoku University School of Medicine, Sendai, Japan. tm30130@mail.cc.tohoku.ac.jp
Yokoyama
Hisayuki
H
Minegishi
Naoko
N
Sasaki
Takeshi
T
Kaku
Mitsuo
M
Harigae
Hideo
H
D000818:Animals; D001773:Blood Cells / Q000166:cytology / Q000378:metabolism*; D015152:Blotting, Northern; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D014157:Transcription Factors / Q000096:biosynthesis*; D014162:Transfection
D014157:Transcription Factors