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Updated: Aug 11, 2026
In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells
Published on: July 24, 2010
Preparation of intact microsomes from cultured mammalian H4IIE cells
Andrey L Sukhodub1, Ann Burchell
1School of Biological Sciences, University of Portsmouth, St. Michaels Building, White Swan Road, Portsmouth, Hampshire, PO1 2DT, England, UK.
Introduction:
Mammalian cell culture is widely used for the cloning and expression of insoluble proteins. The established methods of sub-cellular fractionation of tissues are not always directly suitable for the sub-cellular fractionation of cultured cells. In this study we have optimized the conditions for the preparation of microsomal fractions from cultured cells with the aim of isolating intact vesicles that are suitable for the assay of transport proteins and lumenal enzymes.
Methods:
H4IIE cell cultures were used as a convenient model with high latency of internal endoplasmic reticulum enzyme glucose-6-phosphatase towards mannose-6-phosphate. Also 7-ethoxyresorufin O-deethylase (EROD) activity was determined as a reflection of the state of monooxygenase system.
Results:
The variations in a number of homogenization strokes and buffer composition revealed that one homogenization stroke in glass homogenizer with 0.25 M sucrose, 5 mM HEPES, pH 7.4 buffer provides the best latency/activity ratio for homogenates, but for the isolation of microsomes the higher number of strokes (10) as well as low-osmotic buffer (5 mM HEPES, pH 7.4) are needed. However EROD activity is largely reduced in the preparations using buffers containing sucrose, so 5 mM HEPES buffer is recommended as the most suitable to study the microsomal reactions in H4IIE cells.
Discussion:
The isolation of microsomes was followed by the significant proteolytic breakdown of the glucose-6-phosphatase enzyme. It is recommended to use cell culture homogenates for assays when possible.
Insights
Optimizing microsomal fraction isolation from cultured cells is crucial for studying enzyme activity. Researchers found that specific homogenization and buffer conditions are necessary for intact vesicles, but recommend using cell homogenates for enzyme assays due to protein degradation.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Mammalian cell cultures are vital for expressing proteins, but sub-cellular fractionation differs from tissue methods.
- Optimizing cultured cell fractionation is needed for isolating intact vesicles for enzyme and protein assays.
Purpose of the Study:
- To optimize conditions for preparing microsomal fractions from cultured cells.
- To isolate intact vesicles suitable for assaying transport proteins and lumenal enzymes.
Main Methods:
- Utilized H4IIE cell cultures as a model system.
- Assessed glucose-6-phosphatase latency and 7-ethoxyresorufin O-deethylase (EROD) activity.
- Varied homogenization strokes and buffer compositions (sucrose, HEPES) for microsomal isolation.
Main Results:
- One homogenization stroke in 0.25 M sucrose/HEPES buffer yielded optimal homogenate latency/activity.
- Microsome isolation required 10 homogenization strokes and a low-osmotic buffer (5 mM HEPES).
- Sucrose-containing buffers reduced EROD activity; 5 mM HEPES buffer is recommended for microsomal reactions.
Conclusions:
- Microsome isolation led to significant proteolytic breakdown of glucose-6-phosphatase.
- Cell culture homogenates are recommended for enzyme assays when possible to avoid degradation.
