Determination of optimal conditions for the immobilization of cells in a cell capture enzyme immunoassay (CC-EIA) by a simple Geimsa assay.
To determine the optimal conditions for the immobilization of cells in a cell capture enzyme immunoassay (CC-EIA), the most suitable diluent, and the optimal pH, temperature and period of incubation were examined using WI-38, a human embryonic lung fibroblast cell line. For the evaluation, we devised a simple Giemsa assay method, in which immobilized cells on a microplate were stained with Giemsa solution, the stained dye was eluted with ethanol after washing the plate, and the optimal density (O.D.) was measured at wavelength 620 nm. The optimal conditions for the immobilization were determined to be treatment with 5% formalin in phosphate-buffered saline (PBS) (pH 7.2) for 15 minutes at room temperature, which were confirmed to be suitable for the measurement of cell associated collagen by CC-EIA. Additionally, we found that the simple Giemsa staining method was also useful for evaluating the number of immobilized cells on the microplate after CC-EIA.
Kobayashi
M
M
Department of Ophthalmology, Osaka Medical College, Takatsuki-shi, Japan.
Sano
K
K
Katsumura
K
K
Tanaka
K
K
Sugiyama
T
T
Doi
M
M
Abe
M
M
Ikeda
T
T
D001399:Azure Stains; D002452:Cell Count / Q000379:methods*; D002478:Cells, Cultured; D018914:Cells, Immobilized; D003094:Collagen / Q000032:analysis*; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D007124:Immunoenzyme Techniques; D008168:Lung / Q000166:cytology
D001399:Azure Stains; D003094:Collagen