Fluorescence elisa reader

Activities of the effector Caspases-3 and 7, which play a central role in apoptotic events were evaluated in intestinal tissue samples before and after perfusion using rhodamine based fluorometric assays (Apo-One homogeneous Caspase-3/7 assay, Promega Corporation, Madison, WI, USA). Treatment of the samples and evaluation of Caspase-3/7 activity were done on the basis of the manufacturer’s protocol using a fluorescence ELISA reader (Tecan, Crailsheim, Germany) in combination with the Magellan software v1.1. Protein extraction was performed by using RIPA buffer containing 150 mM sodium chloride, 1 % NP-40, 1 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS) and 50 mM Tris–HCl (pH 7.6; all from Sigma-Aldrich, Hamburg, Germany). The protein concentrations were determined with Roti^®-Quant assays (Carl Roth, Karlsruhe, Germany).

To evaluate the apoptotic activity in the intestinal tissue samples, the activity of the effector Caspases 3 and 7 were measured by using a rhodamine based fluorometric assay (Apo-One homogeneous Caspase-3/7 assay, Promega Corporation, Madison, USA). For evaluation of the Caspase activity, 10 µg of protein concentrate were treated and analysed on the basis of the manufacturer’s protocol using a fluorescence ELISA reader (Tecan, Crailsheim, Austria) in combination with the Magellan software v1.1.

Activities of the effector Caspases-3 and -7, which play a central role in apoptotic events were evaluated in luminal effluent samples before and after perfusion using rhodamine based fluorometric assays (Apo-One homogeneous Caspase-3/7 assay, Promega Corporation, Madison, WI, USA). Treatment of the samples and evaluation of Caspase-3/7 activity were done based on the manufacturer’s protocol using a fluorescence ELISA reader (Tecan, Crailsheim, Austria) in combination with the Magellan software v1.1. Protein extraction was performed with RIPA buffer containing 150 mM sodium chloride, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) and 50 mM Tris-HCl (pH 7.6; all from Sigma-Aldrich, Munich, Germany). Protein concentrations were determined with Roti®-Quant assays (Carl Roth, Karlsruhe, Germany) [13 (link)]. To visualize apoptotic cell nucleus in intestinal tissue sections, Tunel staining was performed by using a apoptosis detection kit (ApopTag Peroxidase In Situ, Merk, Darmstadt, Germany) and DAB substrate (Liquid DAB + Substrate Chromogen System, Dako, CA, USA) as described by the manufacturer.