System for quantitation of gene expression in single cells by computerized microimaging: application to c-fos expression after microinjection of anti-casein kinase II antibody.
A system which allows sensitive and fast automated analysis of weakly labeled fluorescent specimens is described. It is tested in the analysis of c-fos expression stimulated by fetal calf serum and calibrated by quantitation of defined solutions injected into cells with the automated microinjection system. Low light level imaging technology combined with quantitative image processing methods and computer control of the hardware allows fully automated analysis of fluorescent molecules in single living or fixed cells. Reliable methods for subtraction of fluorescent background and automated identification of objects of interest in double-stained cells are described. The accuracy of quantitation is considerably improved by normalizing the fluorescence intensities of respective fluorophores in the same object by the method of ratio imaging. The error rate in determining the relative protein content in single cells is less than 15%. The method is applied to microinjection studies with a monoclonal antibody against casein kinase II subunit beta. Microinjection of this antibody into synchronized cells specifically inhibits c-fos expression stimulated by fetal calf serum. In combination with the computer-automated capillary microinjection system, the technique will become a useful tool in experiments requiring quantitative single cell analysis.
Pepperkok
R
R
Biochemical Instrumentation Program, European Molecular Biology Laboratory, Heidelberg, Germany.
Herr
S
S
Lorenz
P
P
Pyerin
W
W
Ansorge
W
W
D000906:Antibodies / Q000494:pharmacology; D047390:Casein Kinase II; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000187:drug effects; D016762:Genes, fos / Q000187:drug effects; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation*; D008845:Microinjections / Q000295:instrumentation* / Q000379:methods; D017346:Protein Serine-Threonine Kinases / Q000276:immunology; D016760:Proto-Oncogene Proteins c-fos / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000906:Antibodies; D016760:Proto-Oncogene Proteins c-fos; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases