-1::1
Simple Hit Counter
Skip to content

Products

Solutions

×
×
Sign In

CN

EN - EnglishCN - 简体中文DE - DeutschES - EspañolKR - 한국어IT - ItalianoFR - FrançaisPT - Português do BrasilPL - PolskiHE - עִבְרִיתRU - РусскийJA - 日本語TR - TürkçeAR - العربية
Sign In Start Free Trial

RESEARCH

JoVE Journal

Peer reviewed scientific video journal

Behavior
Biochemistry
Bioengineering
Biology
Cancer Research
Chemistry
Developmental Biology
View All
JoVE Encyclopedia of Experiments

Video encyclopedia of advanced research methods

Biological Techniques
Biology
Cancer Research
Immunology
Neuroscience
Microbiology
JoVE Visualize

Visualizing science through experiment videos

EDUCATION

JoVE Core

Video textbooks for undergraduate courses

Analytical Chemistry
Anatomy and Physiology
Biology
Calculus
Cell Biology
Chemistry
Civil Engineering
Electrical Engineering
View All
JoVE Science Education

Visual demonstrations of key scientific experiments

Advanced Biology
Basic Biology
Chemistry
View All
JoVE Lab Manual

Videos of experiments for undergraduate lab courses

Biology
Chemistry

BUSINESS

JoVE Business

Video textbooks for business education

Accounting
Finance
Macroeconomics
Marketing
Microeconomics

OTHERS

JoVE Quiz

Interactive video based quizzes for formative assessments

Authors

Teaching Faculty

Librarians

K12 Schools

Biopharma

Products

RESEARCH

JoVE Journal

Peer reviewed scientific video journal

JoVE Encyclopedia of Experiments

Video encyclopedia of advanced research methods

JoVE Visualize

Visualizing science through experiment videos

EDUCATION

JoVE Core

Video textbooks for undergraduates

JoVE Science Education

Visual demonstrations of key scientific experiments

JoVE Lab Manual

Videos of experiments for undergraduate lab courses

BUSINESS

JoVE Business

Video textbooks for business education

OTHERS

JoVE Quiz

Interactive video based quizzes for formative assessments

Solutions

Authors
Teaching Faculty
Librarians
<<<<<<< HEAD
K12 Schools
Biopharma
=======
K12 Schools
>>>>>>> dee1fd4 (fixed header link)

Language

zh_CN

EN

English

CN

简体中文

DE

Deutsch

ES

Español

KR

한국어

IT

Italiano

FR

Français

PT

Português do Brasil

PL

Polski

HE

עִבְרִית

RU

Русский

JA

日本語

TR

Türkçe

AR

العربية

    Menu

    JoVE Journal

    Behavior

    Biochemistry

    Bioengineering

    Biology

    Cancer Research

    Chemistry

    Developmental Biology

    Engineering

    Environment

    Genetics

    Immunology and Infection

    Medicine

    Neuroscience

    Menu

    JoVE Encyclopedia of Experiments

    Biological Techniques

    Biology

    Cancer Research

    Immunology

    Neuroscience

    Microbiology

    Menu

    JoVE Core

    Analytical Chemistry

    Anatomy and Physiology

    Biology

    Calculus

    Cell Biology

    Chemistry

    Civil Engineering

    Electrical Engineering

    Introduction to Psychology

    Mechanical Engineering

    Medical-Surgical Nursing

    View All

    Menu

    JoVE Science Education

    Advanced Biology

    Basic Biology

    Chemistry

    Clinical Skills

    Engineering

    Environmental Sciences

    Physics

    Psychology

    View All

    Menu

    JoVE Lab Manual

    Biology

    Chemistry

    Menu

    JoVE Business

    Accounting

    Finance

    Macroeconomics

    Marketing

    Microeconomics

Start Free Trial
Loading...
Home
JoVE Journal
Biology
测量重组透气溶性细胞中线粒体底物通量 O-透化细胞
测量重组透气溶性细胞中线粒体底物通量 O-透化细胞
JoVE Journal
Biology
A subscription to JoVE is required to view this content.  Sign in or start your free trial.
JoVE Journal Biology
Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

测量重组透气溶性细胞中线粒体底物通量 O-透化细胞

Full Text
2,779 Views
06:17 min
August 13, 2021

DOI: 10.3791/62902-v

Moustafa Elkalaf1,2, Karolína Vaněčková1,2, Pavla Staňková1, Zuzana Červinková1, Jan Polák*2,3, Otto Kučera*1

1Department of Physiology, Faculty of Medicine in Hradec Králové,Charles University, 2Department of Pathophysiology, Third Faculty of Medicine,Charles University, 3Department of Internal Medicine,University Hospital Kralovske Vinohrady

AI Banner

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study presents a modified protocol for assessing mitochondrial respiratory substrate flux using recombinant perfringolysin O in conjunction with microplate-based respirometry. The research highlights the impact of metformin on mitochondrial respiration across two distinct tumor cell lines, A549 and Hep G2.

Key Study Components

Research Area

  • Mitochondrial metabolism
  • Cellular response to pharmacological treatments
  • Oncology and cancer research

Background

  • The importance of mitochondrial function in various pathologies
  • How drugs like metformin influence cancer cell metabolism
  • Previous methods to assess mitochondrial respiration

Methods Used

  • Microplate-based respirometry
  • A549 and Hep G2 tumor cell lines
  • Recombinant perfringolysin O for permeabilization

Main Results

  • Metformin treatment increased succinate-induced respiration in A549 cells compared to Hep G2 cells.
  • Similar enhancements observed in pyruvate malate and glutamate malate induced respiration.
  • The findings validate the potential of utilizing mitochondrial respiration assays to explore drug impacts in cancer research.

Conclusions

  • The study demonstrates a novel method for evaluating drug effects on mitochondrial function in cancer cells.
  • This could facilitate further research into metabolic targeting in oncology.

Frequently Asked Questions

What are the advantages of using microplate respirometry?
Microplate respirometry allows for high-throughput screening, requiring minimal samples while providing reliable data on mitochondrial function.
How does metformin affect mitochondrial respiration?
Metformin alters mitochondrial respiration, which can reveal important insights into the metabolic adaptation of cancer cells.
What cell lines were used in this study?
A549 and Hep G2 tumor cell lines were analyzed for their mitochondrial respiration responses to metformin treatment.
Why is recombinant perfringolysin O important in this protocol?
Recombinant perfringolysin O is used to permeabilize cell membranes, facilitating the assessment of mitochondrial function without the interference of cellular metabolic processes.
What was a key finding regarding mitochondrial respiration between the two cell lines?
A549 cells exhibited a higher rate of mitochondrial respiration induced by succinate when compared to Hep G2 cells.
Can this method be applied to other types of cancer research?
Yes, this method is versatile and can be adapted to study various cancer types and other conditions affecting mitochondrial metabolism.
How is the assay prepared before measurement?
The assay involves cell seeding, treatment with substrates, and calibration of the respirometry analyzer to ensure accurate results.

在这项工作中,我们描述了一种改进的方案,以使用重组产气溶酶O与基于微孔板的呼吸测定法一起测试线粒体呼吸底物通量。通过该协议,我们展示了二甲双胍如何影响两种不同肿瘤细胞系的线粒体呼吸。

因为它测试线粒体底物通量,这些通量可能会受到不同病理或治疗的影响。例如,今天我们将用它来揭示癌细胞对二甲双胍治疗的反应。它需要最少数量的细胞,同时对每种不同的材料进行足够的重复和适当的控制。

该分析仪仅用于研究用途。该技术可以识别线粒体代谢中的错误,并可用于评估影响线粒体的战士药物。演示该程序的将是Karolina Vaneckova,她是我实验室的本科生和研究技术人员。

以每孔20, 000个细胞的密度开始接种A549细胞,在色谱柱中加入2至11个海马XF 96细胞培养微孔板。将第 1 列和第 12 列留空为背景井。用等体积的细胞培养基填充空白孔,然后将细胞在37摄氏度下在加湿的培养箱中孵育,其中含有5%二氧化碳用于细胞附着。

三到四小时后,在孔中加入100微升细胞培养基。用一毫摩尔二甲双胍处理实验组第7至11柱,用等体积的无菌蒸馏水处理对照组,然后将板返回培养箱。为了给传感器补水,将每口井200微升无菌水移入公用设施板中。

然后小心地将传感器盒放回,同时将传感器浸入水中。将墨盒在37摄氏度的无二氧化碳培养箱中孵育至第二天。打开分析仪和控制单元。

启动仪器控制和数据采集软件,并按照文本手稿中所述设计测定方案。在组定义下,创建四个注入策略,其中端口 A 根据注入的底物而不同,并在底物或缩写之后命名策略。将寡霉素分配到端口B,FCCP分配到端口C,将鱼藤酮抗霉素A混合物分配到端口D.创建并命名八个组,在板图下,将组分配给相应的孔。

然后将协议另存为即用型模板。保持分析仪开关打开,使温度在一夜之间稳定下来。第二天,丢弃公用设施板中的水,并在公用设施板中每孔添加200微升预热的校准剂。

将墨盒返回到无二氧化碳培养箱,直到测定时间。保持培养箱内的湿度源,关闭或将风扇速度降至最低,以避免校准剂快速蒸发。用预热的两次线粒体测定溶液,5%BSA和无菌水制备5毫升底物和抑制剂的工作溶液,如文本手稿中所述。

接下来,按照税务手稿中所述,在注射器端口中加载底物和抑制剂。要在运行检测选项卡下进行校准,请单击开始运行以开始检测。插入加载的审查盒并等待校准完成。

通过将两倍线粒体测定溶液,无菌水和5%BSA混合在50毫升管中,制备20毫升测定培养基。加入两微升10微摩尔重组产气溶素O,以达到1纳摩尔的浓度。并通过轻柔移液重新悬浮混合物,避免摇晃和涡旋。

将管子在37摄氏度下孵育直至使用。使用多通道移液器洗涤细胞和空空白孔两次,使用预热的无钙和镁PBS溶液。丢弃PBS并加入180微升预热的测定培养基以进行细胞透化。

通透后,立即将校准传感器盒的实用程序板替换为含有透化细胞的细胞板并开始测量。治疗组琥珀酸盐诱导呼吸的发生率较高。A549细胞对二甲双胍治疗的反应高于Hep G2。对于丙酮酸苹果酸盐诱导的呼吸,与Hep G2细胞相比,A549细胞在5至15分钟之间显示出增加的诱导作用。

谷氨酸苹果酸盐诱导呼吸和棕榈酰肉碱苹果酸盐诱导呼吸的结果相似。达到合适的底物和抑制剂浓度。必须将正确的卷加载到正确的端口中。

当确定某种底物的代谢途径发生变化时,有必要评估参与该途径的酶和转运蛋白的功能。

View the full transcript and gain access to thousands of scientific videos

Sign In Start Free Trial

Explore More Videos

生物学 第174期 微孔板呼吸测定法 线粒体 二甲双胍 透化细胞 透气溶性蛋白O 线粒体底物

Related Videos

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

13:42

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

Related Videos

22K Views

氧通量测量以评估活细胞和透化细胞中的线粒体呼吸

05:44

氧通量测量以评估活细胞和透化细胞中的线粒体呼吸

Related Videos

296 Views

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

10:39

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

Related Videos

14.2K Views

高分辨率呼​​吸测量,以评估线粒体功能的通透和完整细胞

08:33

高分辨率呼​​吸测量,以评估线粒体功能的通透和完整细胞

Related Videos

42.6K Views

用最小组织量测定果蝇透纤维中线粒体耗氧量

14:55

用最小组织量测定果蝇透纤维中线粒体耗氧量

Related Videos

12.4K Views

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

08:19

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

Related Videos

2.8K Views

实时测量中性粒细胞的线粒体生物能量特征

09:39

实时测量中性粒细胞的线粒体生物能量特征

Related Videos

2.5K Views

测量哺乳动物线粒体功能的氧非依赖性测定

05:59

测量哺乳动物线粒体功能的氧非依赖性测定

Related Videos

3.4K Views

通过高分辨率呼吸测定法测量人和小鼠骨骼肌纤维中的线粒体呼吸

08:12

通过高分辨率呼吸测定法测量人和小鼠骨骼肌纤维中的线粒体呼吸

Related Videos

2.4K Views

高分辨率荧光肺活测定法评估人免疫细胞中线粒体膜电位的动态变化

07:18

高分辨率荧光肺活测定法评估人免疫细胞中线粒体膜电位的动态变化

Related Videos

1.6K Views

JoVE logo
Contact Us Recommend to Library
Research
  • JoVE Journal
  • JoVE Encyclopedia of Experiments
  • JoVE Visualize
Business
  • JoVE Business
Education
  • JoVE Core
  • JoVE Science Education
  • JoVE Lab Manual
  • JoVE Quizzes
Solutions
  • Authors
  • Teaching Faculty
  • Librarians
  • K12 Schools
  • Biopharma
About JoVE
  • Overview
  • Leadership
Others
  • JoVE Newsletters
  • JoVE Help Center
  • Blogs
  • JoVE Newsroom
  • Site Maps
Contact Us Recommend to Library
JoVE logo

Copyright © 2026 MyJoVE Corporation. All rights reserved

Privacy Terms of Use Policies
WeChat QR code