For DNA laddering, apoptotic DNA fragments were extracted according to the methods of Herrmann et al.17 and electrophoresed at 70 V in a 2.0% agarose
gel in Tris-acetate-EDTA buffer. This method of DNA extraction selectively isolates apoptotic, fragmented DNA and leaves behind the intact chromatin. The gel was stained with ethidium bromide and photographed under ultraviolet (UV) illumination. DNA ladder markers (100 basepairs) were added to a lane of each gel as a reference for the analysis of internucleosomal DNA fragmentation. Intact small intestinal crypts were isolated with the distended intestinal sac method as described by Traber et al.18 with slight modifications. Small intestine (jejunum and ileum) was removed and rinsed thoroughly with intestinal wash solution (0.15 M NaCl, 1 mM dithiothreitol [DTT], and 40 Bioactive Compound Library nmr pg/mL phenylmethylsulfonyl fluoride [PMSF]) and then filled with buffer A (in mM): 96 NaCl, 27 sodium citrate, 1.5 KCl, 8 KH2P04, 5.6 Na2HP04, and 40 pg/mL PMSF (pH 7.4). The ends were clamped with microclips and the intestine was filled to a pressure of 50 cm H20. The filled intestine was submerged in oxygenated 0.15 M NaCl at 37°C for 40 minutes, drained, and the solution was discarded. IWR-1 The intestine was then filled with buffer B (in mM): 109 NaCl, 2.4 KCl, 1.5 KH2PO4, 4.3
Na2HPO4, 1.5 EDTA, 10 glucose, 5 glutamine, 0.5 DTT, and 40 pg/mL PMSF (pH 7.4), incubated at 37°C for another 20 minutes, and the intestinal contents were drained and collected. The cells from PIK3C2G 40-60 minutes fraction containing intact and isolated crypts were collected by pelleting at 100g for 5 minutes at 4°C and washed once with PBS. LCM of individual Paneth cells was performed with the PixCell I LCM System (Arcturus Engineering, Mountain View, CA) as described.19 Small intestine tissues were excised and embedded in Optimum Cutting Temperature (OCT) compound (Sakura, Torrance, CA), sectioned
at a thickness of 10 μm, and mounted on 1.0 PEN Membrane Slides (Carl Zeiss, Thornwood, NY). The sections were then prepared for microdissection using an LCM staining kit (Ambion, Austin, TX) through a graded alcohol series (95%, 75%, 50%) followed by cresyl violet staining. After destaining by way of second graded alcohol series (50%, 75%, 95%), they were dehydrated in 100% ethanol followed by xylene. LCM was performed on a Zeiss Axiovert 200M microscope equipped with PALM RoboSoftware and the total area of tissue collected per slide was tracked and recorded. RNA was isolated from the dissected tissue by following the protocol provided by the RNAqueous-Micro kit (Ambion) by way of column purification. Small intestines were fixed in 4% paraformaldehyde / 3% glutaraldehyde in 10 mM sodium phosphate buffer (pH 7.4) for 48 hours. All samples were postfixed with 1% osmium tetroxide in 100 mM cacodylate buffer (pH 7.4) on ice for 1 hour. Samples were then treated with 0.