Name: HeLa Cell Fixation with Hoechst 33342 and Blocking
Uploaded: 2023-05-05T14:55:00
Description: Watch this Scientific Journal Video about HeLa Cell Fixation with Hoechst 33342 and Blocking at JoVE.com

The video outlines a protocol for fixing HeLa cells in a 384-well plate. It involves rinsing the cells with PBS, adding formaldehyde for fixation, followed by additional rinses and the addition of bovine serum albumin (BSA) for blocking, with specific incubation times in the dark at room temperature.

hela cell fixation with hoechst 33342 and blocking

mtDNA의 합성 및 분포에 대한 고처리량 정량화

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.

저자 스포트라이트: 미토콘드리아 DNA 합성 및 분포의 고처리량 이미지 기반 정량화  

미토콘드리아 DNA 합성 및 분포의 고처리량 이미지 기반 정량화

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 HeLa Cell Fixation with Hoechst 33342 and Blocking at JoVE.com

HeLa Cell Fixation with Hoechst 33342 and Blocking  

Hoechst 33342를 이용한 HeLa 세포 고정 및 차단

HeLa 세포 고정을 시작하기 위해, 마이크로플레이트 세척기를 사용하여 HeLa 세포-함유 384-웰 플레이트를 100 마이크로리터의 PBS로 2회 헹구었다. 두 번째 세척 후 우물에 25 마이크로 리터의 PBS를두고 25 마이크로 리터의 8 % 포름 알데히드 용액을 첨가하십시오. 플레이트를 실온에서 30분 동안 어두운 곳에서 배양합니다.그런 다음 각 웰을 100 마이크로리터의 PBS로 4회 헹구고 마지막 세척 후 웰에 25 마이크로리터의 PBS를 남겨 둡니다. PBS에 25마이크로리터의 6% 소 혈청 알부민(BSA)을 각 웰에 넣고 플레이트를 어두운 곳에서 30분 동안 배양합니다.

Research

JoVE Journal

Biochemistry

HeLa Cell Fixation with Hoechst 33342 and Blocking

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 ...

연구

생화학

Preparation of siRNA Mixture for Mitochondrial DNA Synthesis

미토콘드리아 DNA 합성을 위한 siRNA 혼합물의 제조

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 세포에서 세포 형질주입 및 5-브로모-2&#39;-데옥시유리딘(BrdU) 혼입

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

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

HeLa 세포에서 미토콘드리아 DNA의 5-브로모-2&#39;-데옥시우리딘(BrdU) 표지

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

mtDNA 분포를 모니터링하기 위해 형질주입된 HeLa 세포에 1차 및 2차 항체 추가

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

형질감염된 HeLa 세포에서 mtDNA 및 유전자 발현 수준을 측정하기 위한 정량적 이미지 분석