3 and 6 ng/ml, 10 and 20 ng/ml, 30 and 60 ng/ml or 100 and 200 ng/ml respec tively. MDCK cells were treated for 20 hours in complete DMEM media and then placed in DMEM media contain ing 0. 5% FBS without phenol red for the remaining 4 hours prior to assay. Cell culture plates were http://www.selleckchem.com/products/Imatinib(STI571).html centrifuged for 5 min at 1000 g and 100l of supernatant was trans ferred to an optically clear flat bottom 96 well microtiter plate. LDH activity assay was initiated by addition of 100l substrate and absorbance was measured at 492 nm using a SpectraMax250 Platereader. DMEM containing 0. 5% FBS without phenol red was used as assay medium to determine low control. Additionally, a group of cells was lysed with 2% Triton X100 ten min utes prior to supernatant collection to determine total cel lular LDH activity.

Apoptosis was detected using the DeadEnd Fluorometric TUNEL System. MDCK cells grown to confluency on tissue culture treated coverglasses were placed in a variety of conditions for 24 hours. Cells were then fixed with paraformaldehyde in PBS for 25 minutes then permeabilized in PBS containg 0. 2% Tri tion X 100 for 5 minutes. DNA fragments were labeled with fluorescein UTP using a recombinant terminal deox ynucleotidyl transferase for 1 hour at 37 C. Following six wash steps in 2�� SSC and PBS, nuclei were stained with DAPI. Slides were stored in the dark at 4 C prior to micro scopic analysis using a Nikon 2000E microscope. Transepithelial electrical resistance MDCK cells are seeded on Transwell inserts and grown to confluency. Experiments are preformed on cultures after a minimum of ten days culture.

In all experiments, cytokines and inhibitors were delivered to both the apical and basolateral chambers. Measurements of transepithe lial electrical resistance were made using an EVOM epithelial voltohmmeter with an EndOhm 12 mm meas urement chamber calibrated daily using CaliCell. Transwell inserts are transferred to the measurement chamber containing media . the apical electrode is positioned prior to obtaining measurement. Readings taken at time 0 hrs were obtained immediately following addition of drug treatments. The resistance of the epithelium was deter mined by passing a bipolar current across the epithelium and measuring the resultant voltage change. The resist ance of the fluid and insert only between the voltage measuring electrodes was measured and subtracted from the total resistance.

The transepithelial resistance was automatically determined using Ohms law. Paracellular flux assay MDCK cell monolayers in Transwell inserts were incu bated under different experimental conditions in the pres ence of 0. 2Ci/ml of D mannitol or sodium fluorescein in the apical well. At given times, apical and basal media Carfilzomib was with drawn and radioactivity was counted with a scintillation counter.