After 72h of culture, five independent human hepatocyte cultures (biological replicates) were exposed to two concentrations of APAP (5 and 10 mM) for 24h. APAP was dissolved in culture medium.
Growth protocol
Human hepatocytes were obtained from resection specimens of patients undergoing partial hepatectomy for colorectal metastases in an otherwise normal liver at the University Hospital Maastricht, the Netherlands. Surgery was performed as described before (Dejong and Garden, 2003) and in none of the cases hepatic inflow occlusion was applied during liver transaction. All patients provided written informed consent and the study was approved by the Medical Ethics Committee of the University Hospital Maastricht. Isolation of human hepatocytes from resection specimens was performed according to the method described by LeCluyse et al. (LeCluyse et al., 2005). Hepatocyte preparations with viability greater than 75% were included for further studies. Cell suspensions with viability below 85% were purified using a Percoll gradient, as previously described (LeClusye et al., 2005). Cells were cultured on collagen gel precoated 12-well plates at a density of 6.5 x 10-5 cells per well. Human hepatocyte sandwich cultures were essentially prepared according to the method of Beken et al. (Beken et al., 2004). 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 0.1 µM DEX, 6.25 µg/ml insulin, 6.25 µg/ml transferin, and 6.25 ng/ml selenium (Hamilton et al. 2001). Cultures were incubated 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 72h.
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 five human individuals 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 individual were hybridized against cyanine 3-CTP (Cy3) labeled RNA samples from control hepatocyte cultures of the same individual. 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.
After 72h of culture, five independent human hepatocyte cultures (biological replicates) were exposed to two concentrations of APAP (5 and 10 mM) for 24h. APAP was dissolved in culture medium.
Growth protocol
Human hepatocytes were obtained from resection specimens of patients undergoing partial hepatectomy for colorectal metastases in an otherwise normal liver at the University Hospital Maastricht, the Netherlands. Surgery was performed as described before (Dejong and Garden, 2003) and in none of the cases hepatic inflow occlusion was applied during liver transaction. All patients provided written informed consent and the study was approved by the Medical Ethics Committee of the University Hospital Maastricht. Isolation of human hepatocytes from resection specimens was performed according to the method described by LeCluyse et al. (LeCluyse et al., 2005). Hepatocyte preparations with viability greater than 75% were included for further studies. Cell suspensions with viability below 85% were purified using a Percoll gradient, as previously described (LeClusye et al., 2005). Cells were cultured on collagen gel precoated 12-well plates at a density of 6.5 x 10-5 cells per well. Human hepatocyte sandwich cultures were essentially prepared according to the method of Beken et al. (Beken et al., 2004). 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 0.1 µM DEX, 6.25 µg/ml insulin, 6.25 µg/ml transferin, and 6.25 ng/ml selenium (Hamilton et al. 2001). Cultures were incubated 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 72h.
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 five human individuals 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 individual were hybridized against cyanine 3-CTP (Cy3) labeled RNA samples from control hepatocyte cultures of the same individual. 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 (G4110B 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
none
Data processing
RFlagged 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.
