B response. In addition, different viability parameters as the ATP level

B response. In addition, different viability parameters as the ATP level, the WST-1 conversion, the release of lactate dehydrogenase (LDH) and the transepithelial electrical resistance (TEER) were used to get a comprehensive data set to complete the understanding whether Cry1Ab affects the viability of gastrointestinal cells. Furthermore, the two dimensional differential in gel electrophoresis (2D-DIGE) combined with mass spectrometry was employed to supplement the viability approaches to better characterize the possible interactions of Cry1Ab at the molecular and cellular levels. The identified changes of Hsp70 level caused by Cry1Ab treatment were further verified by Western blotting and enzyme-linked immunosorbent assay (ELISA) using specific antibodies.Evaluation of Cell ViabilityCell proliferation was detected using the Cell Proliferation Reagent WST-1 (Roche). The quantitation of ATP was used as a further indicator of viable cells measured with CellTiter-Glo Luminescent Cell Viability Assay (Promega). The released LDH was determined using the CytoTox-ONETM Homogeneous Membrane Integrity Assay (Promega), a fluorimetric method for measuring the release of LDH from cells with damaged membrane. These methods were performed according to the manufacturer’s instructions and have been described in detail by our group in a previous publication [21]. IPEC-J2 cells were treated for 24 h respectively for 48 h with 500 ng/ml and 1 mg/ml Cry1Ab. For all viability assays valinomycin (500 ng/ml, ,0.556 mg/ml respectively) was used as cell damaging control.Real-time Cell PS-1145 custom synthesis AnalysisThe xCELLigence system (Roche Applied Science and ACEA Biosciences) was used to evaluate cell survival after Cry1Ab treatment. The real-time cell analysis was performed under cell culture conditions (37uC, 5 CO2, 95 humidity) on the E-plate 96 that differs from standard 96-well microtiter plates vastly with its incorporated gold cell sensor arrays in the bottom. The impedance of the sensor electrodes was measured to allow purchase tert-Butylhydroquinone monitoring the physiological behaviour of the cells on the electrodes. In the presence of cells, cells attached to the electrode sensor surfaces will lead to an increase in impedancehttp://www. sciencedirect.com/science/article/pii/S0109564109002887 bbib30#bbib30. Thus, the Real-time xCelligence impedance measurement correlates with changes in cell proliferation, viability, and cytotoxicity. The xCELLigence system was used according to the instructions of the supplier (Roche Applied Science and ACEA Biosciences). First, the optimal seeding concentration was determined. After seeding the respective optimal number of cells (10,000/well) in 100 ml medium to the wells of the E-plate 96, the physiological state of the IPEC-J2 cells was monitored every 30 min by the xCELLigence system. After 4 h, when cells were in log growth phase, the IPECJ-2 cells were exposed to 50 ml of medium containing 1 mg/ml Cry1Ab or 500 nM valinomycin, respectively. Controls received medium only, or medium plus ethanol, respectively. Each experiment was run for 24 h.Materials and Methods Cell CultureThe IPEC-J2 cell line is a non-transformed intestinal cell 23977191 line originally derived from jejunal epithelia, isolated from a neonatal, unsuckled piglet [22]. Cells were grown in Dulbecco’s modified eagle medium (DMEM)/Ham’s F-12 (1:1) supplemented with 10 fetal calf serum (Biochrom, Germany) and maintained in an athmosphere of 5 CO2. For viability measurements IPEC-J2 cells were plated.B response. In addition, different viability parameters as the ATP level, the WST-1 conversion, the release of lactate dehydrogenase (LDH) and the transepithelial electrical resistance (TEER) were used to get a comprehensive data set to complete the understanding whether Cry1Ab affects the viability of gastrointestinal cells. Furthermore, the two dimensional differential in gel electrophoresis (2D-DIGE) combined with mass spectrometry was employed to supplement the viability approaches to better characterize the possible interactions of Cry1Ab at the molecular and cellular levels. The identified changes of Hsp70 level caused by Cry1Ab treatment were further verified by Western blotting and enzyme-linked immunosorbent assay (ELISA) using specific antibodies.Evaluation of Cell ViabilityCell proliferation was detected using the Cell Proliferation Reagent WST-1 (Roche). The quantitation of ATP was used as a further indicator of viable cells measured with CellTiter-Glo Luminescent Cell Viability Assay (Promega). The released LDH was determined using the CytoTox-ONETM Homogeneous Membrane Integrity Assay (Promega), a fluorimetric method for measuring the release of LDH from cells with damaged membrane. These methods were performed according to the manufacturer’s instructions and have been described in detail by our group in a previous publication [21]. IPEC-J2 cells were treated for 24 h respectively for 48 h with 500 ng/ml and 1 mg/ml Cry1Ab. For all viability assays valinomycin (500 ng/ml, ,0.556 mg/ml respectively) was used as cell damaging control.Real-time Cell AnalysisThe xCELLigence system (Roche Applied Science and ACEA Biosciences) was used to evaluate cell survival after Cry1Ab treatment. The real-time cell analysis was performed under cell culture conditions (37uC, 5 CO2, 95 humidity) on the E-plate 96 that differs from standard 96-well microtiter plates vastly with its incorporated gold cell sensor arrays in the bottom. The impedance of the sensor electrodes was measured to allow monitoring the physiological behaviour of the cells on the electrodes. In the presence of cells, cells attached to the electrode sensor surfaces will lead to an increase in impedancehttp://www. sciencedirect.com/science/article/pii/S0109564109002887 bbib30#bbib30. Thus, the Real-time xCelligence impedance measurement correlates with changes in cell proliferation, viability, and cytotoxicity. The xCELLigence system was used according to the instructions of the supplier (Roche Applied Science and ACEA Biosciences). First, the optimal seeding concentration was determined. After seeding the respective optimal number of cells (10,000/well) in 100 ml medium to the wells of the E-plate 96, the physiological state of the IPEC-J2 cells was monitored every 30 min by the xCELLigence system. After 4 h, when cells were in log growth phase, the IPECJ-2 cells were exposed to 50 ml of medium containing 1 mg/ml Cry1Ab or 500 nM valinomycin, respectively. Controls received medium only, or medium plus ethanol, respectively. Each experiment was run for 24 h.Materials and Methods Cell CultureThe IPEC-J2 cell line is a non-transformed intestinal cell 23977191 line originally derived from jejunal epithelia, isolated from a neonatal, unsuckled piglet [22]. Cells were grown in Dulbecco’s modified eagle medium (DMEM)/Ham’s F-12 (1:1) supplemented with 10 fetal calf serum (Biochrom, Germany) and maintained in an athmosphere of 5 CO2. For viability measurements IPEC-J2 cells were plated.