1 M NaH2PO4, 7. 4 pH for 15 min utes. After washing in PBS three times, the cells were incubated with 0. 05% Tween 20 in PBS for 15 minutes. After washing in PBS, the cells were incubated with TdT end labelling cocktail for 60 minutes. Termination buffer was added to stop the reaction. After washing 4 times in PBS, cells were blocked for 20 minutes and stained with avidin fluorescein isothiocyanate solution for 30 minutes. After washing with PBS 3 times, fluorescence plate reader quantified the fluorescence of TUNEL posi tive cells. Fast Activated cell based ELISA assay The level of total and phosphorylated PI3Kp85 Tyr, Akt Ser473, GSK 3B Ser9, and FKHR Thr24 were quantified using Fast Activated Cell based ELISA assays according to the manufacturers protocol.
Briefly, OvCa cells were cultured in 96 well plates 200 ul of culture medium one day prior to manipulation. OvCa cells were treated with 0 or 5 ug/ml of cisplatin, along with no additions or 100 ng/ml of CCL25 plus 1 ug/ml of anti CCR9 or isotype control antibodies for 24 hours. In addition, cells were treated with or without kinase inhibi tors of PI3K, and FAK. Cells were then fixed with 4% formaldehyde at room temperature for 20 minutes, followed by washing with PBS containing 0. 1% Triton X 100. Endogenous per oxidase activity was quenched using 1% H2O2 in wash buffer. The cells were incubated in antibody blocking buf fer, followed by incubations with phospho or total anti PI3Kp85 , or Akt or GSK 3B, FKHR specific primary antibodies. After washing steps, horse raddish peroxidase conjugated antibody was added and cells were incubated for one hour at 25 C.
Subsequently, the plates were developed and chemiluminescence was measured using a Spectramax 2 plate reader. Finally, plates were washed and the number of cells in each well was estimated by crystal violet staining, mea suring absorbance at 595 nm. Relative cell numbers were then used to normalize chemiluminescent readings, and the change in phosphorylation status was calculated by dividing chemiluminscence detected using phospho pro tein specific antibody with that of the total protein spe cific antibody. Statistics The data were compared using a two tailed Students t test and expressed as the mean SE. The results were analyzed using the Stat view II program and were labelled statistically significant if p values were 0. 01.
Results Effects of CCL25 on cisplatin induced growth inhibition Dacomitinib SKOV 3 cells incorporated BrdU at a higher rate than OVCAR 3 cells, which suggested SKOV 3 cells proliferated at a higher rate compared to OVCAR 3 cells. In the absence of cisplatin, CCL25 significantly enhanced BrdU incorporation of OVCAR 3 and SKOV 3 cell lines by 1. 5 fold in comparison to untreated cells. However, when these cells were treated with increasing concentrations of cisplatin, CCL25 protected human OvCa cells from cisplatin mediated growth inhibition.