Male Wistar (Crl: (WI) WU BR), 9-12 weeks of age, 180-250 g
Treatment protocol
After 72h of culture, three independent rat hepatocyte cultures (biological replicates) cultured in two conditions (St. and Mod.) were exposed to two concentrations of APAP (5 and 10 mM) for 24h. APAP was dissolved in culture medium.
Growth protocol
Male Wistar rats (Crl: (WI) WU BR), 9-12 weeks of age, were obtained from Charles River GmbH, Sulzfeld, Germany. During the acclimatization period and until sacrifice, animals were housed individually in macrolon cages with wire tops and sawdust bedding at 22 degrees Celsius and 50-60% humidity. The light cycle was 12 h light and 12 h dark. Feed and tap water were available ad libitum. Animal protocols for hepatocyte isolation were approved by the institutional Animal Ethics Committee. Hepatocytes were isolated acording to a two-step collagenase perfusion technique as described by Seglen (Seglen, 1976) with minor modifications. Hepatocyte preparations with viability greater than 85% as determined by Trypan Blue exclusion were used and cultured on collagen gel precoated 6-well plates at a density of 1.3 x 10-E6 cells per well. Sandwich-cultures of rat hepatocytes were essentially prepared according to the method of Beken (Beken et al., 2004) with culture conditions as described by Kienhuis (Kienhuis et al., 2006, Kienhuis et al., 2007). After attachment for 4 h in attachment medium, dead cells were removed by washing and the upper collagen layer was applied. Thereafter, cells were kept in DMEM containing either standard culture medium (St.) consisting of DMEM supplemented with 0.5 U/ml insulin, 7 ng/ml glucagon, 7.5 µg/ml hydrocortisone hemisuccinate, and 50 µg/ml gentamycin, or modified culture medium (Mod.) consisting of standard culture medium (St.) supplemented with 1 mM phenobarbital (PB), 10 µM dexamethasone (DEX), and 5 µM β-naphthoflavone (β-NF) (Kienhuis et al., 2007). PB was added as a concentrated stock solution in PBS. DEX and β-NF were added as concentrated stock solutions in DMSO. The final DMSO concentration was equalized in all culture media (St. and Mod.) and did not exceed 0.2% (v/v). Cultures were incubated at 37 degrees Celsius in a humidified incubator gassed with 5% CO-2 in air. Medium was changed on a daily basis during a period of 72 h.
Extracted molecule
total RNA
Extraction protocol
After 24h exposure time, culture medium of sandwich-cultured hepatocytes was removed, Trizol was added onto the upper collagen layer and cells were collected. RNA was purified using the RNeasy MinElute kit including an additional DNA digestion step. RNA quality was determined using the Bioanalyzer (Agilent Technologies, Palo Alto, CA). All samples contained intact total RNA with a 28S/18S rRNA ratio > 1.5 and a RIN number > 8. RNA extractions of three rats cultured in two conditions (St. and Mod.) were used for microarray analysis.
Label
Cy5
Label protocol
RNA samples from control hepatocyte cultures and cultures exposed to APAP were labeled with cyanine 5-CTP (Cy5). Cy5 labeled samples from one rat were hybridized against cyanine 3-CTP (Cy3) labeled RNA samples from control hepatocyte cultures of the same rat. As a result, control samples labeled with Cy5 hybridized agains Cy3 samples can be considered a self-self hybridization. Labeling was performed using Agilent's low RNA input fluorescent linear amplification kit followin manufacturer's instruction. Briefly, double-stranded cDNA was synthesized using molony murine leukemia virus-reverse transcriptase (MMLV-RT) with T7 promoter primer, starting with 1 µg of total RNA. Cyanine-labeled cRNA targets were transcribed using T7 RNA Polymerase. The amlified cRNA was purified using RNeasy mini spin columns. Synthesized cRNA products were quantified spectrophotometrically.
Male Wistar (Crl: (WI) WU BR), 9-12 weeks of age, 180-250 g
Treatment protocol
After 72h of culture, three independent rat hepatocyte cultures (biological replicates) cultured in two conditions (St. and Mod.) were exposed to two concentrations of APAP (5 and 10 mM) for 24h. APAP was dissolved in culture medium.
Growth protocol
Male Wistar rats (Crl: (WI) WU BR), 9-12 weeks of age, were obtained from Charles River GmbH, Sulzfeld, Germany. During the acclimatization period and until sacrifice, animals were housed individually in macrolon cages with wire tops and sawdust bedding at 22 degrees Celsius and 50-60% humidity. The light cycle was 12 h light and 12 h dark. Feed and tap water were available ad libitum. Animal protocols for hepatocyte isolation were approved by the institutional Animal Ethics Committee. Hepatocytes were isolated acording to a two-step collagenase perfusion technique as described by Seglen (Seglen, 1976) with minor modifications. Hepatocyte preparations with viability greater than 85% as determined by Trypan Blue exclusion were used and cultured on collagen gel precoated 6-well plates at a density of 1.3 x 10-E6 cells per well. Sandwich-cultures of rat hepatocytes were essentially prepared according to the method of Beken (Beken et al., 2004) with culture conditions as described by Kienhuis (Kienhuis et al., 2006, Kienhuis et al., 2007). After attachment for 4 h in attachment medium, dead cells were removed by washing and the upper collagen layer was applied. Thereafter, cells were kept in DMEM containing either standard culture medium (St.) consisting of DMEM supplemented with 0.5 U/ml insulin, 7 ng/ml glucagon, 7.5 µg/ml hydrocortisone hemisuccinate, and 50 µg/ml gentamycin, or modified culture medium (Mod.) consisting of standard culture medium (St.) supplemented with 1 mM phenobarbital (PB), 10 µM dexamethasone (DEX), and 5 µM β-naphthoflavone (β-NF) (Kienhuis et al., 2007). PB was added as a concentrated stock solution in PBS. DEX and β-NF were added as concentrated stock solutions in DMSO. The final DMSO concentration was equalized in all culture media (St. and Mod.) and did not exceed 0.2% (v/v). Cultures were incubated at 37 degrees Celsius in a humidified incubator gassed with 5% CO-2 in air. Medium was changed on a daily basis during a period of 72 h.
Extracted molecule
total RNA
Extraction protocol
After 24h exposure time, culture medium of sandwich-cultured hepatocytes was removed, Trizol was added onto the upper collagen layer and cells were collected. RNA was purified using the RNeasy MinElute kit including an additional DNA digestion step. RNA quality was determined using the Bioanalyzer (Agilent Technologies, Palo Alto, CA). All samples contained intact total RNA with a 28S/18S rRNA ratio > 1.5 and a RIN number > 8. RNA extractions of three rats cultured in two conditions (St. and Mod.) were used for microarray analysis.
Label
Cy3
Label protocol
RNA samples from control hepatocyte cultures and cultures exposed to APAP were labeled with cyanine 5-CTP (Cy5). Cy5 labeled samples from one rat were hybridized against cyanine 3-CTP (Cy3) labeled RNA samples from control hepatocyte cultures of the same rat. As a result, control samples labeled with Cy5 hybridized agains Cy3 samples can be considered a self-self hybridization. Labeling was performed using Agilent's low RNA input fluorescent linear amplification kit followin manufacturer's instruction. Briefly, double-stranded cDNA was synthesized using molony murine leukemia virus-reverse transcriptase (MMLV-RT) with T7 promoter primer, starting with 1 µg of total RNA. Cyanine-labeled cRNA targets were transcribed using T7 RNA Polymerase. The amlified cRNA was purified using RNeasy mini spin columns. Synthesized cRNA products were quantified spectrophotometrically.
Hybridization protocol
For microarray hybridization, Cy5-labeled samples and Cy3-labeled samples were combined. cRNAs were fragmented at 60 degrees Celsius for 30 min with fragmentation solution followed by hybridization on Agilent 22K format 60-mer oligo microarrays (G4130A from Agilent Technologies, Palo Alto, CA) for 17h at 60 degrees Celsius with Agilent hybridization solution. Arrays were washed according to manufacturer's instruction.
Scan protocol
Microarrays were scanned using a Packard Scanarray Express confocal laser scanner (PerkinElmer, Boston, MA).
Description
St_Standard culture medium
Data processing
Flagged spots, consisting of poor quality spots and negative and positive control spots, were excluded. For each spot, median local background intensity was subtracted from the median spot intensity and spots from low expression genes (with a net intensity of < 40 in both channels), were excluded from further analysis. These background-corrected median intensities were log transformed base 2. Data were normalized using the Lowess algorithm.
