Then, the microrarrays were washed 3 times for 6 min with binding buffer and 3 times for 6 min with MilliQ water in the dark.
After a further incubation period of 90 min at room temperature, filters were washed three times for 30 min with binding buffer.
First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly dI-dC), salmon spoly dI-dCand wild-type DNA (biotinylated, 10 pmol ml−1).
The blot was incubated for 1.5 h with 1 μg/mL biotin-tagged CRD from human mincle in 5% (w/v) BSA in binding buffer, washed 3× 5 min with binding buffer and incubated with 0.5 μg/mL alkaline phosphatase-conjugated extravidin (Sigma) in 5% (w/v) BSA in binding buffer for 1 h at room temperature.
After 3× 5 min washes with binding buffer, the blot was developed for 2 min with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate substrate (Millipore).
The chip was washed 3 times (5 min each) with binding buffer followed by a brief rinse with water.
To perform EMSA binding reaction, 5 μg of nuclear extracts were incubated (10 min, 25 °C) with binding buffer (10 mM Tris, 50 mM NaCl, 1 mM dithiothreitol, 0.1 mM EDTA, 5% (v/ v) glycerol, pH 7.5) containing 0.25 μg poly [d(I C)] and 5 μg bovine serum albumin.
Cells were harvested by centrifugation at 6,000 × g at 4 °C for 15 min, washed with binding buffer (20 mM Tris HCl buffer containing 500 mM NaCl and 5 mM imidazole, pH 7.9) and then suspended in 50 ml of the same buffer.
Briefly, cells were resuspended in binding buffer at 1×106 cells/ml and stained with Annexin V and propridium iodide for 15 min, diluted 1∶1 with binding buffer, filtered, and counted immediately on a FACScalibur flow cytometer.
Pre-treatment (30 min) with the cholesterol binding agent nystatin (30 µg/ml) abolished the apoptogenic effect of DC101 (Figure 2D) at all doses up to 5 µg/ml, significantly reducing apoptosis from 30±2% to 9±1% (p<0.005).
In brief, gel purified 'body' labelled RNA was incubated with tRNA (50 μg/ml) at 65°C for 5 min, renatured at room temperature for 10 min and then mixed with binding buffer (50 mM Tris-HCl pH 7.5, 40 mM KCl, 35 mM NaCl, 25 μg/ml tRNA, 0.5 mM DTT, 50 μg/ml BSA) in a total of 10 μl, and incubated at room temperature for 20 min with either 0, 200, 400 or 600 mM ELAV protein.
