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

Journal

生物化学

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

May 05, 2023

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

01:31 min

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1,242 Views

Preparation of siRNA Mixture for Mitochondrial DNA Synthesis

00:45 min

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

01:19 min

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

01:44 min

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246 Views

HeLa Cell Fixation with Hoechst 33342 and Blocking

01:05 min

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Addition of Primary and Secondary Antibodies to the Transfected HeLa Cells to Monitor the mtDNA Distribution

02:04 min

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Quantitative Image Analysis to Measure the mtDNA and Gene Expression Levels in the Transfected HeLa Cells

03:02 min

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96 Views

Lukasz S. Borowski^1,2, Karolina Kasztelan^2,3, Jolanta Czerwinska-Kostrzewska^1,2, Roman J. Szczesny²

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.