Regeneration and luminescence of aequorin in Chinese hamster ovary cells transformed with cDNA for apoaequorin.
Aequorin, a photoprotein which is regenerated from apoaequorin by incubation with coelenterazine, emits light when it binds Ca2+. The aim of this study was to determine if apoaequorin could be used in adherent mammalian cells for measuring cytosolic Ca2+, and imaging Ca2+, at the single cell level. Chinese hamster ovary (CHO-K1) cells were stably transformed with apoaequorin cDNA and expressed apoaequorin while attached to the culture dishes. Maximal luminescence intensity was obtained when 0.5 x 10(6) cells/ml were grown and incubated with 2.5 microM coelenterazine for 4 hr at 20 degrees C. Ca2+ mobilizing agents (ionomycin and maitotoxin) induced luminescence in CHO-K1 transformed cells. However, imaging of light emission from single cells proved to be unsuccessful. Ca2+ could be readily measured in the adherent CHO-K1 cells, but imaging was not possible at the single cell level.
Sanchez-Bueno
A
A
Osaka Bioscience Institute, Japan.
Yoshida
R
R
Tsuji
F I
FI
D000331:Aequorin / Q000737:chemistry* / Q000235:genetics*; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000378:metabolism; D005260:Female; D015759:Ionomycin / Q000494:pharmacology; D008163:Luminescent Measurements; D008387:Marine Toxins / Q000494:pharmacology; D010053:Ovary / Q000378:metabolism; D035964:Oxocins; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D037401:Scyphozoa; D014162:Transfection; D000095702:Polyether Toxins
D000331:Aequorin; D001059:Apoproteins; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D015759:Ionomycin; D008387:Marine Toxins; D035964:Oxocins; D011994:Recombinant Proteins; C060014:apoaequorin; C020394:maitotoxin; D000095702:Polyether Toxins