Research Paper Volume 7, Issue 11 pp 911—927

Figure 3. Rapamycin treatment of DKC skin fibroblast cultures.

(A) Cumulative population doublings of skin fibroblasts. Fibroblast cultures from the mother I-2, the daughter II-2 and a healthy control were trypsinized and viable cells determined by trypan blue staining. Viable cells remained unstained. Population doublings were calculated using the following equation: PD=X + log2(Y/I) where: X = initial PD I = cell inoculum (number of cells plated in the flask) Y = final cell yield (number of cells at the end of the growth period). Cell were defined as dead (indicated with a cross) if no remaining viable cells were detected. (B) Δ-gal senescence assay of skin fibroblasts. Fibroblasts were cultured in DMEM/10% FCS/1%PenStrep containing either 5nM rapamycin dissolved in DMSO or the equal volume of DMSO as negative control (equivalent to a 1:1,000 dilution). For the indicated timepoints, cells were fixed after 24h with 0.1% glutaraldehyde and stained for Δ-galactosidase (Δ-Gal) at pH 6 as described in Materials and Methods. Nuclei were visualized by staining with DAPI (Sigma-Aldrich), diluted 1:10,000. Coverglasses were embedded in moviol 4-88 (Carl Roth) on slides. Cells were observed at 20-fold magnification and pictures taken at brightfield and fluorescent light with a filter set suitable for DAPI on an Olympus CellR microscope. Depicted overlays of brightfield and fluorescence were merged in ImageJ. Images are representative of the indicated time points. (C) Quantification of Δ-gal assay. Δ-Gal-positive cells were detected at the indicated time points using a programmed plugin for the image editing program ImageJ as described in Materials and Methods. The quantified images were representative of the indicated time points. 200 cells counted by DAPI staining were analyzed for each time point and measurement. The fibroblasts were determined as Δ-Gal positive when blue staining in the brightfield reached a defined intensity and surrounding area of the core that was detected in the fluorescence light (Ex 330-385, Em LP420 filter set for DAPI detection).