Comparison of three methods for mitochondria isolation from the human liver cell line (HepG2).
OBJECTIVE
The aim of this study was to evaluate and compare three available methods for mitochondrial isolation from a human cell line to predict the best method for each probable application.


BACKGROUND
Organelle isolation is gaining importance in experimental laboratory settings. Mitochondrial dysfunction may affect tumorgenesis process. There are some evidences that transplantation of healthy, intact and active mitochondria into cells containing defective mitochondria may reduce the proliferation. Therefore, isolated mitochondria could be considered as an effective tool for assessment and management of mitochondrial related disorders.


METHODS
Mitochondrial isolation from the human liver cell line (HepG2) was performed using two commercially available kits, including Qproteome (Qiagen) and MITOISO2 (Sigma-Aldrich), as well as a manual method. Integrity of inner membrane of mitochondria was assessed by JC-1 staining. Activity of isolated mitochondria was evaluated by DCFH-DA staining, and total yield of isolated mitochondria determined by micro-Lowry method. Finally, relative quantification using Real-time PCR was conducted to compare the mtDNA copy number of mitochondria isolated by three different methods.


RESULTS
Compared to other methods, manual kit resulted in higher yields of total amount of mitochondrial protein and mtDNA copy numbers. Isolated mitochondria by Qproteome kit, showed a higher activity. Finally, the integrity of inner-membrane of isolated mitochondria was significantly higher in Qproteome when compared with the other two methods.


CONCLUSIONS
Due to differences in quality, quantity and activity of isolated mitochondria using three techniques discussed here, the method in which best-suited to each research project should be selected according to the distinct features of isolated mitochondria.
Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
Pedram
P
Azimzadeh
Basic and Molecular Epidemiology of Gastrointestinal Disorders, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Hamid
H
Asadzadeh Aghdaei
Basic and Molecular Epidemiology of Gastrointestinal Disorders, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Peyman
P
Tarban
Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
Mohammad Mahdi
MM
Akhondi
Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
Abolfazl
A
Shirazi
Genetic Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
Hamid Reza
HR
Khorram Khorshid
Cell line; HepG2; Mitochondrial isolation