Name: 5-Bromo-2'-Deoxyuridine (BrdU) Labeling of Mitochondrial DNA in HeLa Cells
Uploaded: 2023-05-05T14:55:00
Description: Watch this Scientific Journal Video about 5-Bromo-2'-Deoxyuridine BrdU Labeling of Mitochondrial DNA in HeLa Cells at JoVE.com

The video explains a method for labeling mitochondria in HeLa cells using BrdU to detect newly synthesized DNA. It details the preparation of a BrdU solution, the addition of a tracking dye, and incubation steps, highlighting the specific detection of BrdU signals in treated cells compared to untreated controls.

5-bromo-2&#39;-deoxyuridine (brdu) labeling of mitochondrial dna in hela cells

mtDNA'nın sentezi ve dağılımının yüksek verimli miktar tayini

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.

Yazar Spotlight: Mitokondriyal DNA Sentezi ve Dağılımının Yüksek Verimli Görüntü Tabanlı Miktar Tayini  

Mitokondriyal DNA Sentezi ve Dağılımının Yüksek Verimli Görüntü Tabanlı Nicelleştirilmesi

The protocol aims to identify proteins with dysfunction leads to changes in the number of mitochondrial DNA molecules. This research may also reveal the factors involving the distribution of mitochondrial DNA within the mitochondria. Mitochondrial replication and maintenance mechanisms are still not fully understood.Much less is known about regulating the distribution of mitochondrial genomes within organoids and the proteins involved in it. As a result, identifying and characterizing some new players will be of great importance. The main advantage of the technique is its high-throughput and high content protocol.We can simultaneously test many experimental conditions and measure various parameters during a single experiment. In genome-wide studies, the proposed protocol provides an opportunity to outline a global picture of how nuclear encoded genetic information regulates its mitochondrial counterpart. Additionally, it has the potential to identify proteins whose dysfunction causes mitochondrial DNA stress and activates the interferon response pathway.

Watch this Scientific Journal Video about 5-Bromo-2'-Deoxyuridine BrdU Labeling of Mitochondrial DNA in HeLa Cells at JoVE.com

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

HeLa hücrelerinde mitokondriyal DNA&#39;nın 5-bromo-2&#39;-deoksiüridin (BrdU) etiketlemesi

Mitokondri etiketlemesine başlamak için, DMEM'de% 10 fetal sığır serumu veya FBS ile 20 mikromolar BrdU çözeltisi hazırlayın. Ardından, son konsantrasyonu 1.1 mikromolara ayarlamak için mitokondri izleme boyasını ekleyin. 15 saat sonra, kuyucuklar içeren HeLa hücre süspansiyonuna 10 mikrolitre mitokondri izleme boya çözeltisi ekleyin.Hücreleri bir saat boyunca inkübe edin. Yeni sentezlenen DNA moleküllerini BrdU ile etiketlemek için kullanılan koşullar, HeLa hücrelerinin mitokondrilerinde BrdU etiketli DNA'nın tespit edilmesine izin verdi. Anti-BrdU antikorları ile elde edilen sinyal spesifikti ve sadece BrdU ile tedavi edilen hücrelerde gözlendi.BrdU tedavisinden bağımsız olarak, sıfır sıra hücrelerin mitokondrilerinde hiçbir anti-BrdU veya anti-DNA antikor sinyali tespit edilmedi. Anti-BrdU antikorlarından gelen floresan sinyalinin ölçülmesi, BrdU ile tedavi edilen sıfır sıra hücrelerinin, BrdU ile tedavi edilmemiş hücrelerle aynı düşük floresan seviyesini gösterdiğini, ebeveyn hatları A549 ve HeLa için sinyalin 50 kat daha yüksek olduğunu göstermiştir.

Research

JoVE Journal

Biochemistry

5-Bromo-2&#39;-Deoxyuridine (BrdU) Labeling of Mitochondrial DNA in HeLa Cells

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 ...

Biyokimya

Preparation of siRNA Mixture for Mitochondrial DNA Synthesis

Mitokondriyal DNA Sentezi için SiRNA Karışımının Hazırlanması

Cell Transfection and 5-Bromo-2&#39;-Deoxyuridine (BrdU) Incorporation in HeLa Cells

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

HeLa hücrelerinde hücre transfeksiyonu ve 5-bromo-2&#39;-deoksiüridin (BrdU) katılımı

HeLa Cell Fixation with Hoechst 33342 and Blocking

Hoechst 33342 ile HeLa Hücre Fiksasyonu ve Blokajı

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

mtDNA Dağılımını İzlemek için Transfekte HeLa Hücrelerine Primer ve Sekonder Antikorların Eklenmesi

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

Transfekte HeLa hücrelerinde mtDNA ve gen ekspresyon seviyelerini ölçmek için kantitatif görüntü analizi