4. ImmunohistochemistryTo identify cellular markers, P3 cells were seeded on poly-L-lysine-coated 8-well selleck kinase inhibitor chamber slides (BD Biosciences), cultured for another 1-2 days and subjected to immunocytochemistry and immunofluorescence staining.4.1. Immunofluorescence StainingSamples were rinsed briefly in PBS, fixed in ice-cold methanol for 10min, and then allowed to dry completely. After permeabilisation with 0.025% Triton X-100 (Merck, Darmstadt, Germany), the cells were incubated with 1.5% normal goat or donkey blocking serum (Santa Cruz Biotechnology) in PBS for 30min at 37��C to suppress nonspecific binding of IgGs. After washing three times with PBS (5min each) the cells were incubated overnight at 4��C with the primary antibodies listed in Table 1.

After three PBS washes, cells were incubated with FITC and Texas-red- (Santa Cruz Biotechnology) labelled appropriate secondary antibodies for 25min in dark. After washing three times with PBS, the cells were mounted with mounting medium containing DAPI (Santa Cruz Biotechnology).Table 1Immunocytochemical properties of hAT-MSCs and hBM-MSCs.4.2. Immunoperoxidase StainingImmunocytochemical analysis was performed using the streptavidin-peroxidase method (UltraVision Plus Large Volume Detection System Anti-Polyvalent, HRP immunostaining Kit, Thermo Scientific, UK). To reduce nonspecific background staining due to endogenous peroxidase, cultured cells were fixed in ice-cold methanol with 0.3% hydrogen peroxide (Carlo Erba Reactifs, Val-De-Reuil Cedex Pa Des Portes, FRANCE) for 15min and allowed to dry.

After additional PBS washes, cells were incubated with Ultra V Block for 5min at room temperature. Then, cells were incubated overnight at 4��C with the primary antibodies listed in Table 1. The following day, cells were incubated with biotinylated secondary antibodies for 15min at room temperature. Incubations were followed by streptavidin peroxidase treatment for 15min at room temperature and signals were detected with the AEC kit (Zymed Laboratories, UK). The cells were counterstained with hematoxylin (Santa Cruz Biotechnology) and examined under a light microscope (Leica DMI 4000B, Wetzlar, Germany). After induction of apoptosis (described below) cells were stained with caspase-3.5. Apoptosis Induction and Detection5.1.

2mmol/L H2O2-Induced ApoptosisAt passage 3 hBM-MSCs and hAT-MSCs (n = 3) were seeded into 6-well plate at a density of 10 �� 104/cm2 and cultured for a further 48h and medium changed to apoptosis inducing Drug_discovery medium that contains 2mmol/L H2O2 MEM ve 10% FBS. After 60min, cells were washed with PBS and apoptotic cell percentage was detected by flow cytometry with Annexin-V-FITC Apoptosis Detection Kit (BD Pharmingen). The data were analyzed with the Cell Quest software (BD Biosciences).5.2.