The video describes a laboratory protocol for staining HeLa cells to visualize mitochondrial DNA using primary and secondary antibodies. It details the steps of incubation, washing, and analysis, highlighting the impact of downregulating mitochondrial transcription factor A (TFAM) on mitochondrial DNA distribution, observed through fluorescence intensities.

addition of primary and secondary antibodies to the transfected hela cells to monitor the mtdna distribution

Cuantificación de alto rendimiento de la síntesis y distribución de ADNmt

For most eukaryotic cells mitochondria are essential organelles, as they play a crucial role in numerous cellular processes. First and foremost, mitochondria are the key energy suppliers of cells1. Mitochondria are also involved in regulating cellular homeostasis (for instance, intracellular redox2 and the calcium balance3), cell signaling4,5, apoptosis6, the synthesis of different biochemical compounds7,8, and the innate immune response9. Mitochondrial dysfunction is associated with various pathological states and human diseases10. 
The functioning of mitochondria depends on the genetic information located in two separate genomes: the nuclear and mitochondrial genomes. The mitochondrial genome encodes a small number of genes compared to the nuclear genome, but all the mtDNA-encoded genes are essential for human life. The mitochondrial protein machinery necessary to maintain the mtDNA is encoded by nDNA. The basic components of the mitochondrial replisome, as well as some mitochondrial biogenesis factors, have already been identified (reviewed in previous research11,12). However, mitochondrial DNA replication and maintenance mechanisms are still far from being understood. In contrast to nDNA, the mitochondrial genome exists in multiple copies, which provides an additional layer for regulating mitochondrial gene expression. Much less is currently known about the distribution and segregation of mtDNA within organelles, to what extent these processes are regulated, and if they are, which proteins are involved13. The segregation pattern is crucial when cells contain a mixed population of wild-type and mutated mtDNA. Their unequal distribution may lead to the generation of cells with a detrimental amount of mutated mtDNA.
So far, the protein factors necessary for mtDNA maintenance have been identified mainly by biochemical methods, bioinformatic analyses, or through disease-associated studies. In this work, in order to ensure a high chance of identifying factors that have previously escaped identification, a different strategy is described. The method is based on the labeling of mtDNA during replication or repair with 5-bromo-2'-deoxyuridine (BrdU), a nucleoside analog of thymidine. BrdU is readily incorporated into nascent DNA strands during DNA synthesis and, in general, is used for monitoring the replication of nuclear DNA14. However, the procedure developed here has been optimized for detecting BrdU incorporated into mtDNA using the immunofluorescence of anti-BrdU antibodies.
The approach allows for the high-throughput quantification of mtDNA synthesis and distribution in human cells cultured in vitro. A high-throughput strategy is necessary to conduct tests under different experimental conditions in a relatively short time; therefore, it is proposed in the protocol to utilize a multi-well format for cell culturing and automated fluorescence microscopy for imaging. The protocol includes the transfection of human HeLa cells with an siRNA library and the subsequent monitoring of mtDNA replication or repair using the metabolic labeling of newly synthesized DNA with BrdU. This approach is combined with immunostaining of the DNA with the help of anti-DNA antibodies. Both parameters are analyzed using quantitative fluorescence microscopy. Additionally, mitochondria are visualized with a specific dye. To demonstrate the specificity of the protocol, BrdU staining was tested on cells devoid of mtDNA (rho0 cells), on HeLa cells upon the silencing of well-known mtDNA maintenance factors, and on HeLa cells after treatment with an mtDNA replication inhibitor. The mtDNA levels were also measured by an independent method, namely qPCR.

Autor destacado: Cuantificación basada en imágenes de alto rendimiento de la síntesis y distribución del ADN mitocondrial  

Cuantificación basada en imágenes de alto rendimiento de la síntesis y distribución del ADN mitocondrial

Watch this Scientific Journal Video about Addition of Primary and Secondary Antibodies to the Transfected HeLa Cells to Monitor the mtDNA Distribution at JoVE.com

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

Adición de anticuerpos primarios y secundarios a las células HeLa transfectadas para monitorear la distribución del ADNmt

Para comenzar la adición del anticuerpo primario, aspire la albúmina sérica bovina o BSA de las células HeLa transfectadas fijas y bloqueadas dejando 10 microlitros de solución BSA en el pozo. Luego agregue 10 microlitros de la solución de anticuerpos primarios preparada en BSA al 3% en PBS e incube la placa en la oscuridad a cuatro grados centígrados durante la noche. Después de la incubación, enjuague cada pocillo cuatro veces con 100 microlitros de PBS, dejando 10 microlitros después del último lavado.A continuación, agregue 10 microlitros de solución de anticuerpos secundarios a la placa de pocillos 384. Use anticuerpos específicos de isotipo conjugados con fluorocromos, como Alexa Fluor 488 y Alexa Fluor 555. Después de incubar la placa en la oscuridad durante una hora, enjuague cada pocillo cuatro veces con 100 microlitros de PBS y deje 50 microlitros de PBS en el pozo después del último lavado.El uso de anticuerpos anti-ADN permitió el monitoreo de la distribución del ADN mitocondrial en la célula bajo las condiciones experimentales dadas. La regulación a la baja del factor de transcripción mitocondrial A TFAM condujo a cambios drásticos en la distribución del ADN mitocondrial con menos manchas de ADN mitocondrial que en las células de control. La cuantificación de la señal fluorescente media del anticuerpo anti-ADN mostró un aumento de varias veces tras el silenciamiento de TFAM en comparación con las otras condiciones probadas.

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Research

JoVE Journal

Biochemistry

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

108 vistas. Para comenzar la adición del anticuerpo primario, aspire la albúmina sérica bovina o BSA de las células HeLa transfectadas fijas y bloqueadas dejando 10 microlitros de solución BSA en el pozo. Luego agregue 10 microlitros de la solución de anticuerpos primarios preparada en BSA al 3% en PBS e incube la placa en la oscuridad a cuatro grados centígrados durante la noche. Después de la incubación, enjuague cada pocillo cuatro veces con 100 microlitros de PBS, dejando 10 microlitros desp...

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

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

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

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.

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...