Development of electrochemical reporter assay using HeLa cells transfected with vector plasmids encoding various responsive elements.
Electrochemical assay using HeLa cell lines transfected with various plasmid vectors encoding SEAP (secreted alkaline phosphatase) as the reporter has been performed by using SECM (scanning electrochemical microscopy). The plasmid vector contains different responsive elements that include GRE (glucocorticoid response elements), CRE (cAMP responsive elements), or kappaB (binding site for NFkappaB (nuclear factor kappa B)) upstream of the SEAP sequence. The transfected HeLa cells were patterned on a culture dish in a 4x4 array of circles of diameter 300 microm by using the PDMS (poly(dimethylsiloxane)) stencil technique. The cellular array was first exposed to 100 ng mL(-1) dexamethasone, 10 ng mL(-1) forskolin, or 100 ng mL(-1) TNF-alpha (tumor necrosis factor alpha) after which it was further cultured in an RPMI culture medium for 6 h. After incubation, the cellular array was soaked in a measuring solution containing 4.7 mM PAPP (p-aminophenylphosphate) at pH 9.5, following which electrochemical measurements were performed immediately within 40 min. The SECM method allows parallel evaluation of different cell lines transfected with pGRE-SEAP, pCRE-SEAP, and pNFkappaB-SEAP patterned on the same solid support for detection of the oxidation current of PAP (p-aminophenol) flux produced from only 300 HeLa cells in each stencil pattern. The results of the SECM method were highly sensitive as compared to those obtained from the conventional CL (chemiluminescence) protocol with at least 5x10(4) cells per well.
Shiku
Hitoshi
H
Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579, Japan. shiku@bioinfo.che.tohoku.ac.jp
Takeda
Michiaki
M
Murata
Tatsuya
T
Akiba
Uichi
U
Hamada
Fumio
F
Matsue
Tomokazu
T
D000469:Alkaline Phosphatase / Q000235:genetics; D002452:Cell Count; D004563:Electrochemistry; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D049449:Luminescence; D008853:Microscopy; D010957:Plasmids / Q000235:genetics*; D020218:Response Elements / Q000235:genetics*; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D014162:Transfection
D000469:Alkaline Phosphatase