The cellular viability was monitored after 3, 5 and 7 days of culture with the Cell Counting Kit-8 (CCK-8, Fluka) assay.
In order to ensure that the assay output was within the linear range we first determined the plating density for our specific cell type.
3T3 cells showed a linear correlation within a range of 1600–150,000 cells/ well.
The total number of cells at confluence was counted with a hemocytometer and found to be ca. 300,000 cells/well.
The plates were incubated at 37 °C in 5% CO2 for 2 h to allow the CCK-8 reaction to occur, as per manufacturer instructions.
The media with the kit were removed to a 96-well Elisa plate; fresh medium was used as control.
The plate was placed into a pectrophotometric plate reader (Elisa reader) set to read the absorbance at 450 nm, which directly correlates to the number of living cells in the culture.