Microdismembrator

Liquid nitrogen-frozen tissue was homogenized using a micro-dismembrator (Sartorius, Göttingen, Germany). RNA extraction from mouse tissue was performed using the Roti Quick kit (Carl Roth, Karlsruhe, Germany) followed by RNA purification with the peqGold RNA isolation kit (Peqlab, Erlangen, Germany), according to the manufacturers’ instructions. For RNA quality assessment, RNA integrity numbers (RIN) were measured via Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and samples with RIN number above 6.5 were used for sequencing. RNA library preparation was done using the Illumina TruSeq RNA Sample Prep kit v2 following the manufacturers’ protocol. RNA sequencing was performed as 100-bp paired-end runs on a HiSeq2500 (Illumina). Pools of 16 indexed libraries were sequenced on four lanes. Image analysis and base calling were performed using Illumina Real Time Analysis. CASAVA 1.8 was used for demultiplexing.

Samples, i.e. UV-irradiated material and non-irradiated control samples of all species, were directly filtered after removal from the chamber through pre-weighted glass fiber filters (Whatman GF/C; VWR) using a vacuum pump. For each petri dish 6 filters were used. The filters were freeze-dried (Heto power dry PL 6000, Thermo Fisher Scientific, Waltham, USA) for 48 h and their dry weight was determined. The dried filters were ground in a Micro-Dismembrator (Sartorious) in pre-cooled Teflon jars and extracted with methanol/water (25:75) in an ultrasonic bath (Bandelin Sonorex 35 kHz, Berlin, Germany) for 15 min at 45 °C. After centrifugation at 3000 rpm for 10 min, the supernatant was collected and evaporated at 45 °C in a rotary vacuum evaporator (Büchi, Flawil, Switzerland). For storage at −20 °C these extracts were transferred in screw-cap glasses and again lyophilized. For HPLC analysis solutions with a concentration of 10 mg/ml were prepared in water.

Cholesterol, triglyceride and free fatty acid contents were analyzed in the plasma and quadriceps muscle isolated from mice after three weeks of treatment. Muscle samples were homogenized with a microdismembrator for 30 s at 3000 rpm (Sartorius Stedim Biotech, Aubagne, France). For the cholesterol and triglyceride contents, muscles were lysed in lysis buffer from the assay kit (ab65390 and ab178780, Abcam, Cambridge, UK) and measured according to the manufacturer’s instructions. For the free fatty acid content, muscles were lyzed in chloroform containing 1% Triton X, centrifuged at 4 °C for 5 min at 16,000× g and the supernatant was vacuum-dried at 50 °C for 30 min. Dried samples were reconstituted and measured according to the manufacturer’s instructions (ab65341, Abcam, Cambridge, UK). Cholesterol, triglyceride and free fatty acid contents were measured directly from the plasma according to the manufacturer’s instructions.

NC-rich NP tissue was harvested from the IVDs of porcine donors (n = 5, ~3 months old). The tissue was lyophilized (Labconco, Kansas City, MO, USA) overnight, resulting in a dry and brittle matrix, which was subsequently pulverized using a microdismembrator (Sartorius, Goetingen, Germany). The NCM powder was aliquoted and stored at −80 °C until further use.

In another set of experiments, ∼3 × 10⁶ cells/animal were injected intraportally for tumor growth in both liver and lungs. For the pancreas, ∼1.5×10⁶ cells/animal were injected orthotopically. After four weeks of tumor growth, animals were sacrificed and tumor nodules re-isolated from these organs for total RNA isolation and histological evaluation.
Total RNA was extracted as detailed in the Qiagen RNA isolation kit. Briefly, tissues weighing ∼100 mg were taken from each sample and disrupted in liquid nitrogen using a microdismembrator (Sartorius, GmbH, Germany) at 2500 vibrations/1 min. The powdered samples were homogenized with 5ml Qiazol lysis reagent and allowed to incubate at room temperature for 5 min. Genomic DNA was depleted by mixing samples with 500 μl of genomic DNAs solution. Total RNA was extracted with chloroform and washed with 70% ethanol. Total RNA was subsequently separated with RNeasy columns using RWT and RPE buffers. The concentrations of isolated RNA were determined by Nano drop spectrophotometer (NanoDrop Technologies, Wilmington, DE). In the core facility, the quality of RNA was evaluated by gel analysis using the total RNA Nano chip assay on an Agilent 2100 Bioanalyzer (Agilent Technologies GmbH, Germany).

To detect the enzymatic activity of MMPs within the anastomosis, gelatin zymography was performed as previously described [22 (link), 26 (link)]. Briefly the anastomotic tissue and non- operated normal murine colon samples as controls, were rapidly frozen in liquid nitrogen and ground using a micro-dismembrator (Sartorius, Göttingen, Germany). The resulting powder was reconstituted in ice-cold NaCl buffer (50 mM Tris/HCl [pH7.5], 75 mM NaCl) containing 1 mM phenylmethylsufonyl fluoride. Samples were centrifuged at 4°C for 20 minutes at 13,000g. Total amounts of protein were determined from the supernatants using a protein assay kit (Bio-Rad, München, Germany). 1 μg of total protein was mixed with 2 x SDS buffer without reducing agent and then added to an 8% SDS gel containing1g/l gelatin. Following electrophoresis the gel was washed using 2.5% Triton X-100 for 30 min at room temperature on a shaking rack. The Zymogram was then incubated overnight at 37°C in a zymogram developing buffer (50mM Tris, 0,2 M NaCl, 5 mM CaCl₂, 0,02% Brij 35). Subsequently staining with Coomassie Blue R-250 was performed for 60 min. For destaining 45% methanol/10% acetic acid was used. Gelatinase activity appear as light bands in the dark blue background of the stained gel.