Iranian J Parasitol: Vol. 6, No.2, 2011, pp.9-16 Non-adherent cells (mainly lymphocytes) were removed and counted in a hemacytometer after staining with trypan blue dye. Lymphocytes (1-2×106 cells/well) were cultured on a 24-well plate, containing 1 ml of 15% heat-inactived fetal bovine se- rum and RPMI-1640 medium in pH 7.4 at 37°C, in a humidified atmosphere of 5% CO2 for 24 hours. The viability of the cells was checked again as before. After verification of the cell viability, different volumes of hydatid fluid including 25 μl, 50 μl, 100 μl, 150 μl and 200 μl were added to each well containing 1.8 ml media respectively. After six times test 200 μl of hydatid fluid (fertile and infer- tile) as inducer lymphocyte (optimum vol- ume) were added to each well containing 1.8 ml media. Additional wells without hydatid fluid (HF) were used as controls to compare with HF-treated lymphocytes. Optimum vol- umes were determined with observation of HF-treated lymphocytes by inverted micro- scope after 12 hours. Inverted microscope revealed that 200 μl of hydatid fluid was optimum volume for lymphocyte shrinkage and apoptotic-body formation. The exposure time to hydatid fluid for evaluation of Cas- pase-3 activity and the mRNA expression levels of Bax and Bcl-2 genes was deter- mined according to the recommendations of a study by Jafari et al. (21). After six hours of exposure to hydatid fluid (Fig. 1), lymphocytes were separated by cen- trifugation at 10000g for 1 min. Caspase-3 activity was determined by the Caspase- 3/CPP32 Fluorometric Assay Kit (K105) (Biovision Inc., MountainView, California, USA) as manufacturer's manual. Activity was measured by a Fluorometer (Jasco, FP- 6200, Japan), as enzyme activity converts a blue emission (λ =404 nm) to a yellow-green color (λ =505 nm). Intensity was defined per milligram of total protein contents of the solubilized cells (according to total protein 11 assessment, measured by spectrophotometer (λ =280 nm). The mRNA expression levels of Bax and Bcl-2 genes were analyzed by semi-quantita- tive reverse transcriptase PCR (RT-PCR) after 12 hours of exposure of lymphocytes with hydatid fluid. For this purpose, lympho- cytes from fertile and infertile HF-treated wells as well as controls were separated by centrifugation at 13000g for 1 min, and pel- lets were used for RNA extraction by Tri- Pure Isolation Reagent (Roche, Germany) according to the manufacturer instructions. Total RNA was reverse transcribed (RT) into cDNA using MBI Revert Aid (Fermen- tase, LifeSciences, Lithuania). A RT-PCR was carried out to determine the mRNA ex- pressionlevels of Bcl-2, and Bax genes. The RT-PCR was performed in a 20-µl reaction volume containing 0.5 µL of cDNA, 1.5 mM of Mgcl2, 125 µM of dNTP, 0.1 U/ µL and 0.5 pmol of specific primers. All the primers used in RT-PCR were designed by Gen Run- ner & Primer Premier Software (Table 1). GAPDH (Glyceraldehydes-3-phosphate deh- ydrogenase) mRNA was used as internal control to adjust the amount of mRNA in each sample. The samples were denaturized for 5 min at 95 °C, and amplified using 35 cycles of 95 °C for 30 s, 60 °C for 80 s, and72 °C for 45 s for Bcl-2 and Bcl-x genes followed by a final elongation at 72 °C for 3 min on a Corbett Research thermocycler (Sydney, Australia). The optimal numbers of cycles were selected for amplification of all three genes experimentally, to be sure that the reactions were in the exponential range of amplification. Five µL of the amplifica- tion product analyzed by electrophoreses on a 2% ethidium-stained agarose gel and docu- mented by a gel documentation system (Syn- gene, U.K.). Quantifications of the PCR band intensities were accomplished by Ko- dak 1D image analysis software (Eastman Kodak Co). The values were normalized by the value obtained from the GAPDH mRNA.