So far no one has described a protocol on how to use Atlantic cod hepatocytes in bioassays, mainly due to the high fat content in these cells, causing the cells to burst during isolation. In this work, we were able to isolate intact liver cells from one mature female individual. The hepatocytes were...
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| Language: | English |
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| Online Access: | http://citeseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.541.7006 http://www.gene-quantification.de/qpcr2007/P095-qPCR-2007.pdf |
| Summary: | So far no one has described a protocol on how to use Atlantic cod hepatocytes in bioassays, mainly due to the high fat content in these cells, causing the cells to burst during isolation. In this work, we were able to isolate intact liver cells from one mature female individual. The hepatocytes were exposed to different concentrations of PCB 138, which is one of the most widespread PCB congeners and of major concern in the marine environment (AMAP, 1998). Three potential reference genes and six target genes were examined with real-time RT-PCR analyses. The results showed that heat shock cognate 70 was slightly more stable than EF1A and β-actin. We were not able to quantify significant differences between the control and the exposed groups for CYP1A, Vtg, GSH-Px and GR. Further only minor, non-significant differences were found between the control group and the groups of cells exposed to PCB 138 for GST and Mn SOD. The study suggest that both β-actin and EF1A are suitable reference genes in qPCR studies of cod hepatocytes. Reference gene stability in hepatocyte primary cell cultures of Atlantic cod (Gadus morhua) Methods Primary cultures of Atlantic cod hepatocyes were isolated from one adult mature female individual (3.0 kg). The hepatocytes were isolated with a modified version of a method previously described by Bell and coworkers (Bell et al., 1997). The hepatocytes had a viability of 80 % and cells were plated on a culture plates (3,8 cm2/well) with a density of 3,5 x 106 per well. After 24 h the hepatocytes were exposed for 24 h to |
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