High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

Lukasz S. Borowski; Karolina Kasztelan; Jolanta Czerwinska-Kostrzewska; Roman J. Szczesny

doi:10.3791/65236

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

May 05, 2023

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution (Video) | JoVE

01:31 min

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Preparation of siRNA Mixture for Mitochondrial DNA Synthesis (Video) | JoVE

00:45 min

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Cell Transfection and 5-Bromo-2'-Deoxyuridine (BrdU) Incorporation in HeLa Cells (Video) | JoVE

01:19 min

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5-Bromo-2'-Deoxyuridine (BrdU) Labeling of Mitochondrial DNA in HeLa Cells (Video) | JoVE

01:44 min

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HeLa Cell Fixation with Hoechst 33342 and Blocking (Video) | JoVE

01:05 min

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Addition of Primary and Secondary Antibodies to the Transfected HeLa Cells to Monitor the mtDNA Distribution (Video) | JoVE

02:04 min

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Quantitative Image Analysis to Measure the mtDNA and Gene Expression Levels in the Transfected HeLa Cells (Video) | JoVE

03:02 min

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Lukasz S. Borowski², Karolina Kasztelan³, Jolanta Czerwinska-Kostrzewska², Roman J. Szczesny

¹Faculty of Biology, Institute of Genetics and Biotechnology,University of Warsaw, ²Institute of Biochemistry and Biophysics,Polish Academy of Sciences, ³International Institute of Molecular and Cell Biology in Warsaw

Summary

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.