EZ4U - CELL PROLIFERATION ASSAY
|Application:||Reduction of tetrazolium salt to coloured formazan|
|Sample type:||culture medium|
|Sample size:||200 µl / test|
|Supplied as:||10 x 96 tests|
The assay set-up is performed in a manner similar to the standard 3H-thymidine incorporation method. Instead of pulsing with tritiated nucleotide, 20 μl of dye solution is added to 200 μl sample. Incubation time is dependent on the metabolic capacity of the cells. Usually 2 to 5 hours of incubation at 37°C are sufficient to yield a significant increase in colour intensity. As different cells vary in their ability to convert the yellow coloured tetrazolium compound to its red formazan derivative, we recommend testing every new cell-line's metabolic capacity as described in Fig.2. After incubation, the plate is removed from the incubator and gently mixed by tipping the plate at all 4 sides. To avoid increased standard deviations, the plate must be shaken before reading the optical density.
The absorbance is measured by a microplate-reader, set at 450 nm or 492 nm with 620 nm as a reference. The reference absorbance at 620 nm (or any wavelength between 620-690 nm) is used to correct for nonspecific background values, caused by cell debris, fingerprints, or other potential interferences. However, the reference may be omitted without significant changes in the accuracy of the assay.
Fig. 2. Different metabolic capacity of various cell lines.
3x103 cells/well were cultivated in 200 μl RPMI1640. Following a cultivation period of 3 days, 25 μl of the dye substrate were added to each well. Optical density was recorded after 4 hours, showing significant differences in the metabolic capacity of the various cell lines.