Quantitative detection of cell surface protein expression by time-resolved fluorimetry.
A method is introduced for quantitative detection of cell surface protein expression. The method is based on immunocytochemistry, the use of long decay time europium(III) chelate and platinum(II) porphyrin labels, and detection of photoluminescence emission from adhered cells by time-resolved fluorimetry. After immunocytochemistry, the assay wells are evaporated to dryness and measured in the dry state. This protocol allows repeated and postponed analysis and microscopy imaging. In order to investigate the performance of the method, we chose expression of intercellular adhesion molecule-1 (ICAM-1) of endothelial cell line EAhy926 as a research target. The expression of ICAM-1 on the cells was enhanced by introduction of a cytokine, tumour necrosis factor-alpha (TNFalpha). The method gave signal:background ratios (S:B) of 20 and 9 for europium and platinum labels, respectively, whereas prompt fluorescent FITC label gave a S:B of 3. Screening window coefficients (=Z'-factor) were >0.5 for all the three labels, thus indicating a score for an excellent screening assay. In conclusion, the method appears to be an appropriate choice for protein expression analysis, both in high-throughput screening applications, and for detailed sample investigation by fluorescent microscopy imaging.
Huttunen
Roope J
RJ
Laboratory of Biophysics, Department of Anatomy, and Medicity Research Laboratories, Institute of Biomedicine, University of Turku, Turku, Finland.
O'Riordan
Tomás C
TC
Härkönen
Pirkko L
PL
Soini
Juhani T
JT
Meltola
Niko J
NJ
Hänninen
Pekka E
PE
Soini
Aleksi E
AE
D002460:Cell Line; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1