Cell viability was confirmed and proliferation was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega Corp, Madison, Wis). These data were analyzed with a one-way analysis of variance and post hoc Bonferroni multiple comparisons test. Cell viability and proliferation data were further used to normalize data derived from the interleukin data analysis. Statistical significance was defined as P<.05.
Conditioned media samples were obtained from six replicate wells in all experimental strain profiles. These samples were then frozen at –80°C (–112°F) until assayed. The cytokine array membranes (RayBiotech Inc, Norcross, Ga) were first incubated with blocking buffer at room temperature (22°C [72°F]) for 30 minutes and then incubated for 4 additional hours at room temperature with 2.5 mL of thawed conditioned media from each of the baseline and treatment groups. The membranes were next incubated overnight at 4.0°C (39.2°F) in primary biotin-conjugated antibody, followed by incubation with horseradish peroxidase-conjugated streptavidin at room temperature for 30 minutes. Finally, the membranes were briefly incubated with detection buffer, exposed to film, and developed using a Kodak X-OMAT 1000A processor (Eastman Kodak Company, Rochester, NY).
After the films were scanned (ScanJet 6200C; Hewlett Packard Company, Palo Alto, Calif), the volume (ie, color density × spot area) of each representative cytokine spot was quantified using AlphaEaseFC software (Version 4.0.0; Alpha Innotech Corporation, San Leandro, Calif). Each cytokine spot was assayed in duplicate for each membrane probed. Intraspot variability for any specific interleukin that was assayed in this manner typically averaged less than 20%. Varying background shades of the film were corrected with the AlphaEaseFC software in the final volume calculations of each duplicate cytokine spot.
Cytokine data were further analyzed with RayBio Cytokine Antibody Array software (Human Cytokine Antibody Array System VI & 6.1; RayBiotech Inc, Norcross, Ga), which was used to average duplicate data for each individual membrane. Corrected cytokine spot volume data, which were normalized with cellular proliferation data, were then analyzed using Microsoft Excel software (Version 11.0; Microsoft Corporation, Redmond, Wash) and GraphPad Prism software (GraphPad Prism 4.03; GraphPad Software Inc, San Diego, Calif).
To determine the effects the different strain profiles had on fibroblast interleukin secretion, the fold changes (ΔF
treatment) of the spot volumes were calculated by comparing each experimental cytokine (E) with the cytokine's baseline cell secretion (BCS) using the following equation:
\[{\Delta}\mathrm{F}_{\mathrm{treatment}}=\mathrm{E}{\div}\mathrm{BCS}\]
Therefore, ΔF
treatment < 1 corresponded to condition media containing less interleukin than at baseline, whereas ΔF
treatment > 1 corresponded to increased interleukin secretion compared with baseline. In keeping with literature standards,
18,19 ΔF
treatment ≥ 2 corresponded to notably increased interleukins as a result of strain.
For the conditioned media collected from each of the strain profiles, each interleukin of interest, as determined by the results of the cytokine protein array, were further analyzed using a human enzyme-linked immunosorbent assay (ELISA) kit. The interleukins tested with individual ELISAs included IL-1α, IL-1β, IL-1ra, IL-2, IL-4, and IL-7 (ELH-IL1α-001, ELH-IL1β-001, ELH-IL1ra-001, ELH-IL2-001, ELH-IL4-001, ELH-IL7-001, respectively; RayBiotech Inc, Norcross, Ga); IL-3 and IL-16 (KHC0031, KHC0161, respectively; BioSource International Inc, Camarillo, Calif); and IL-6 (EH2IL6; Pierce Biotechnology Inc, Rockford, Ill). In addition, ELISA data were normalized with cellular proliferation data and analyzed with two-tailed, two-sample t tests. Statistical significance was defined as P<.05.