Splenocyte Effect on Fibroblast Collagen

To determine the impact of splenocytes on collagen production in fibroblasts, splenocytes of the different experimental groups were co-cultured with murine C4 fibroblasts. According to Van Linthout et al. (11 (link)), fibroblasts were plated at a density of 10,000 cells per well in Iscove Basal Medium (Sigma-Aldrich) containing 10% FBS and 1% penicillin/streptomycin (both Biochrom). Twenty-four hours later, isolated splenocytes were added at a ratio of 1 to 10 (fibroblasts to splenocytes) in Iscove medium (Sigma) supplemented with 10% FBS and 1% penicillin/streptomycin (both Biochrom) in the presence of 50 ng/ml of PMA and 500 ng/ml of Ionomycin (both BD Biosciences). After 24-h co-culture, splenocytes were removed, and fibroblasts were fixed with cold methanol. Subsequently, Sirius red staining was performed. For photometric analyses at 540 nm, the Spectra Max 340PC microplate reader (Molecular Device GmbH) was used.