Reducing background cytokine expression in epithelial cells without serum starvation

Graphical abstract


Immune response assay
RPMI 1640 was aspirated from the wells and cells were gently washed twice with PBS. 350 ml of media no 1-4 was added.
Cells were then incubated at 37 8C, 95% humidity. Supernatants were collected at 0 h and 3 h and stored at À20 8C.

Cytokine analysis
Cell supernatants were analysed using the Cytometric Bead Array System (BD, Bioscience, Sweden) for IL-6 and IL-8 following the manufacturers procedure. Samples were analysed using a BD FACSCanto II flow cytometer (BD Bioscience, Sweden).

Viability assay
2 Â 10 4 A498 cells were seeded in a 96-well plate as described above. After overnight incubation, RPMI 1640 medium was removed and cells were gently washed twice with PBS. 100 ml of media 1-4 were added in triplicates. Cells were then incubated at 37 8C, 95% humidity for 3 h. Subsequently, the LIVE/DEAD 1 Viability/Cytotoxicity Kit (Molecular Probes 1 , Invitrogen, Sweden) was performed according to the manufacturer's instructions. Briefly, cells were incubated with 5 mM ethidium homodimer-1 and 0.5 mM Calcein for 20 min at 37 8C. As a positive control, cells were treated with saponin 0.2% for 10 min prior to LIVE/DEAD stain application, which corresponded to 100% death. Wells with only media 1-4 and LIVE/DEAD stain were used to subtract background fluorescence from the media. To determine viability, cells were excited at 494 nm and fluorescence was detected at 517 nm in a microplate reader (TECAN, Switzerland) to estimate % dead cells or they were observed by epi-fluorescence microscopy (Nikon, Sweden) using a gfp and cy3 filter.

Data analysis
Data were analysed using the FCAP Array software Version 3.0 (BD Biosciences, Sweden). Final graphs and statistical analysis were performed using Prism 6 (GraphPad Software, USA). Statistical significance was determined using Welch's ttest. Statistical significance was determined to be P 0.05.

Results
Cells cultivated in media containing various FBS preparations demonstrated different levels of inflammatory responses as measured by IL-6 and IL-8 expression (Fig. 1). FBS containing >0.25 EU/ml of endotoxin and Performance Plus FBS which contains less than 0.30 EU/ml of endotoxin induced significant levels of IL-6 and IL-8 expression compared to serum starved cells. Cells incubated in media with FBS which had been charcoal stripped did not express significant levels of IL-6 or IL-8. Cells incubated with charcoal stripped FBS have comparable viability to other FBS preparations, while serum starvation led to an increased percentage of cell death (Fig. 2). This data indicates that this charcoal stripped serum can be used as a media additive in experiments to study the inflammatory response without the need to serum starve the cells.