Dataset Information

Fluorescence based cell counting in collagen monolayer cultures of primary hepatocytes.

ABSTRACT: Accurate determination of cell number is essential for the quantitative description of biological processes. The changes should be related to a measurable reference e.g. in the case of cell culture, the viable cell number is a very valuable reference parameter. Indirect methods of cell number/viability measurements may have up to 10 % standard deviation. This can lead to undesirable large deviations in the analysis of "-omics" data as well as time course studies. Such data should be preferably normalized to the exact viable cell number at a given time to allow meaningful interpretation and understanding of the biological processes. Manual counting of cell number is very laborious and not possible in certain experimental setups. We therefore, developed a simple and reliable fluorescence based method with an accuracy of 95-98 % for the determination of the viable cell number in situ. We optimized the seeding cell densities for primary rat hepatocytes for optimal cell adhesion. This will help in efficient use of primary cells which are usually limited in availability. The method will be very useful in the application of "-omics" techniques, especially metabolome analysis where the specific rates of uptake/production of metabolites can be reliably calculated.

SUBMITTER: Priesnitz C

PROVIDER: S-EPMC4960120 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Fluorescence based cell counting in collagen monolayer cultures of primary hepatocytes.

Priesnitz C C Sperber S S Garg R R Orsini M M Noor F F

Cytotechnology 20141126 4

Accurate determination of cell number is essential for the quantitative description of biological processes. The changes should be related to a measurable reference e.g. in the case of cell culture, the viable cell number is a very valuable reference parameter. Indirect methods of cell number/viability measurements may have up to 10 % standard deviation. This can lead to undesirable large deviations in the analysis of "-omics" data as well as time course studies. Such data should be preferably n ...[more]

PMID: 25424145

Similar Datasets

Project description:Liver-enriched transcription factors (LETF) play a crucial role in the control of liver-specific gene expression and for hepatocytes to retain their molecular and cellular functions complex interactions with extra cellular matrix (ECM) are required However, during cell isolation ECM interactions are disrupted and for hepatocytes to regain metabolic competency cells are cultured on ECM substrata. The regulation of LETFs in hepatocytes cultured on different ECM has not been studied in detail. We therefore compared two common sources of ECM and evaluated cellular morphology and hepatocyte differentiation by investigating DNA binding activity of LETFs at gene specific promoters and marker genes of hepatic metabolism. Furthermore, we studied testosterone metabolism and albumin synthesis to assess the metabolic competence of cell cultures. Despite significant difference in morphological appearance and except for HNF1β (p<0.001) most LETFs and several of their target genes did not differ in transcript expression after Bonferroni adjustment when cultured on collagen or Matrigel. Nonetheless, Western blotting revealed HNF1β, HNF3α, HNF3γ, HNF4α, HNF6 and the smaller subunits of C/EBPα and C/EBPβ to be more abundant on Matrigel cultured cells. Likewise, DNA binding activity of HNF3α, HNF3β, HNF4α, HNF6 and gene expression of hepatic lineage markers were increased on Matrigel cultured hepatocytes. To further investigate hepatic gene regulation, the effects of Aroclor 1254 treatment, e.g. a potent inducer of xenobiotic defense were studied in vivo and in vitro. The gene expression of C/EBP-α increased in rat liver and hepatocytes cultured on collagen and this treatment induced DNA binding activity of HNF4α, C/EBPα and C/EBPβ and gene expression of CYP1A1 and CYP1A2 in vivo and in vitro. Taken collectively, two sources of ECM greatly affected hepatocyte morphology, activity of liver enriched transcription factors, hepatic gene expression and metabolic competency that should be considered when used in cell biology studies and drug toxicity testing.

Dataset Information

Fluorescence based cell counting in collagen monolayer cultures of primary hepatocytes.

Publications

Fluorescence based cell counting in collagen monolayer cultures of primary hepatocytes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets