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Sep 2018

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A New Efficient Method for Measuring Oxygen Consumption Rate Directly ex vivo in Human Epidermal Biopsies
一种直接离体测量人体表皮活检耗氧率的新方法   

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Abstract

Skin cells are constantly exposed to environmental influences such as air pollution, chemicals, pathogens and UV radiation. UV radiation can damage different biological structures, but most importantly cellular DNA. Mitochondria contain their own genome and accumulate UV-induced DNA mutations to a large extent. This can result, e.g., in accelerated skin aging. Understanding the impact of harmful external influences on mitochondrial function is therefore essential for a better view on the development of age-related diseases. Previous studies have been carried out on cell cultures derived from primary cells, which does not fully represent the real situation in the skin, while the mitochondrial parameters were considered barely or not at all. Here we describe a method to measure mitochondrial respiratory parameters in epithelial tissue derived from human skin biopsies using an Agilent Seahorse XF24 Flux Analyzer. Before the assay, epidermis and dermis are separated enzymatically, we then used the XF24 Islet capture microplates to position the epidermis samples to measure oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). In these plates, small nets can be fixed to the plate bottom. The epidermis was placed with the vital–basal–side on the net. Active ingredients in the three ports were injected consecutively to determine the effect of each compound. This allows determining the efficiency of the individual complexes within the respiratory chain. This protocol enables the testing of toxic substances and their influence on the mitochondrial respiration parameters in human epithelial tissue.

Keywords: Oxygen consumption rate (耗氧率), Mitochondrial respiration (线粒体呼吸), Epidermis (表皮), Ageing (老化), Skin biopsies (皮肤活检), Ex vivo (离体)

Background

The human skin is the largest organ of the human body and functions as a physical barrier shielding the body from a number of harmful external agents such as air pollution or solar radiation (Gebhard et al., 2014). Solar radiation, e.g., induces increased production of reactive oxygen species (ROS) and thereby DNA damage and mutations in skin, which results in skin aging and skin cancer (Gebhard et al., 2014). Mitochondrial dysfunction resulting from DNA damage is thought to play an important role in these and other essential cellular processes. The accumulation of reactive oxygen species (ROS)-damaged mitochondrial DNA (mtDNA) and proteins can induce mitochondrial dysfunction within the electron transport chain (ETC) and in turn lead to an enhanced ROS production and increased mitochondrial dysfunction (Furda et al., 2012; Yoshida et al., 2012). According to the ‘mitochondrial theory of aging’, this vicious cycle is a major cause for cellular aging, tissue dysfunction and degeneration (Harman, 1972). Understanding the impact of harmful external influences on mitochondrial function is therefore essential for a better view on aging in general. Unfortunately, the measurement of mitochondrial respiration in cell cultures cannot completely reflect the real situation in skin. We therefore established an efficient method to measure the mitochondrial respiration ex vivo directly in human epidermis biopsies with the Seahorse XF24 Flux Analyzer and Seahorse XF24 Islet Capture microplates. The XF24 Analyzer is a multi-well plate system which measures oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) by changes in the fluorescence of solid state fluorophores. In this system, up to four different ports can be used to inject compounds to obtain a mitochondrial respiration profile. The procedure described in this protocol includes the preparation of the biopsies, the preparation of the epidermis for insertion into the XF24 Analyzer and the measurement of mitochondrial parameters by the addition of specific mitochondrial inhibitors. This protocol was established for the testing of toxic substances and their influence on the mitochondrial respiration parameters in human epithelial tissue. In addition, age-related effects of various substances can be tested based on the age of the donor. The analysis of mitochondrial respiration in epidermis derived from skin biopsies represents an important addition to existing screenings.

Materials and Reagents

  1. Centrifuge tubes 15, 50 ml (SARSTEDT, catalog numbers: 62.554.001, 62.547.254)
  2. Microscope slides (Carl Roth, catalog number: 1879.1)
  3. Petri dishes (SARSTEDT, catalog number: 83.3902)
  4. Pipette tips 10 μl, 20 μl, 200 μl, 1,000 μl (SARSTEDT, catalog numbers: 70.1130, 70.116, 70.760.002, 70.762)
  5. Sterile filter 0.2 μm (SARSTEDT, catalog number: 83.1826.001)
  6. Human skin biopsies (kept in 1x PBS at 4 °C for a maximum of 36 h)
  7. 2-Propanol (Carl Roth, catalog number: 9866.1)
  8. Dispase II (neutral protease, grade II) (Roche, catalog number: 04942078001, storage temp. 4 °C)
  9. Dulbecco’s modified Eagle’s medium, high glucose (Sigma-Aldrich, catalog number: D7777-10L, storage temp. 4 °C)
  10. KBMTM Gold Keratinocyte Growth Medium BulletKitTM (Lonza, catalog number: 00192060, storage temp. 4 °C)
  11. KCl (Carl Roth, catalog number: 6781.3)
  12. KH2PO4 (Carl Roth, catalog number: 3904.2)
  13. Na2HPO4·12H2O (Carl Roth, catalog number: N350.1)
  14. NaCl (Carl Roth, catalog number: 3957.3)
  15. NaOH (Carl Roth, catalog number: 6771.3)
  16. Seahorse XF Calibrant Solution (Agilent Technologies, catalog number: 100840-000)
  17. Seahorse XF Cell Mito Stress Test Kit (Oligomycin, FCCP, Rotenone/Antimycin A) (Agilent Technologies, catalog number: 103015-100, storage temp. -20 °C)
  18. 10 M NaOH (see Recipes)
  19. 1x PBS (see Recipes)
  20. Enzymatic digestion Solution (see Recipes)
  21. XF assay medium (see Recipes)

Equipment

  1. Scissor, stainless steel (Carl Roth, catalog number: HCX4.1)
  2. Seahorse Capture Screen Insert Tool (Agilent Technologies, catalog number: 101135-100)
  3. Seahorse XF24 Islet Capture FluxPak containing sensor cartridges and Islet Capture Microplates (Agilent Technologies, catalog number: 101174-100)
  4. Tweezers, stainless steel (Carl Roth, catalog number: 2687.1)
  5. Incubator at 37 °C without CO2 (GFL, catalog number: 4010)
  6. pH Meter FiveEasyTM F20 (Mettler Toledo, catalog number: 30266626)
  7. Pipettes, Eppendorf Research® Plus 10 μl, 20 μl, 200 μl, 1,000 μl (Eppendorf, catalog numbers: 3123000020, 3123000039, 3123000055, 3123000063)
  8. Seahorse XF24 Flux Analyzer (Agilent Technologies, model: XF24, catalog number: 100737-100)

Software

  1. Seahorse XF24 Flux Analyzer Software (instrument software) (Agilent Technologies, Version 1.8.1.1)
  2. Wave (analysis software) (Agilent Technologies, Version 2.6)

Procedure

Day before the assay

  1. Take a biopsy from the sample tube and wash first in 70% isopropanol followed by 1x PBS to remove any residues of blood and other impurities.
  2. In the next step, remove fat and tissue (see Figures 1A and 1B).
  3. Cut the biopsy (see Figure 1B) into small pieces (0.4 x 0.4 cm approximately) (see Figure 1C) and place them in enzymatic digestion solution in a 15 ml centrifuge tube.
  4. Store the biopsy for enzymatic digestion at 4 °C for 16 h (under non-stirring conditions). 
  5. Hydrate the sensor cartridge in Seahorse XF Calibrant according to the manufacturer’s instructions one day prior to measurement in a non-CO2 incubator.
  6. Turn on the XF24 analyzer to preheat the system to 37 °C.


    Figure 1. Preparation steps of the biopsy. A. Fat and connective tissue adherent to the dermis/epidermis (arrow). B. Dermis/Epidermis after first purification step. C. Divided biopsy in solution for enzymatic digestion. D. Separated epidermis sheets after incubation with Dispase II. E. Capture screen insert tool with Islet capture screens (Agilent Technologies).

Day of the assay
  1. Prepare the XF assay medium and heat to 37 °C. 
  2. After enzymatic digestion, carefully remove epidermis from the dermis (tweezers) and place in Petri dish with 1x PBS (see Figure 1D). In this step, it is important to ensure that the basal side is facing down towards the bottom of the petri dish. The epidermal sheet should be placed with the vital–basal–side on the capture screen. This orientation is essential for the measurement as the side facing the capture screen gets detected by the sensor (see Figure 2E). 
  3. Carefully lift the epidermis from the 1x PBS with a microscope slide and wipe it off on a second slide (turn over the epidermis once).
  4. Wipe off the skin with the second slide on the capture screen, pull it smoothly and insert the capture screen with the capture screen insert tool into the Islet capture microplate (see Figure 1E). The epidermis should cover the entire surface of the capture screen to ensure that it’s always to measure the same epidermis area (see Figures 2A-2C). There should be no air bubbles (see Figure 2D) under the epidermis, as they interfere with the measurement.
  5. Remove excess overhanging skin in the well with tweezers.
  6. Add 450 μl XF assay medium into each well of the Islet capture microplate.
  7. Incubate the Islet capture microplate in the incubator (without CO2) at 37 ° C for 45 min to adjust the epidermis to the new medium.
  8. Load the injection ports of the sensor cartridge as programmed (see the programming of the XF controller), follow the instructions of the XF Analyzer and start the calibration of the sensor cartridge.
  9. When calibration is complete, place the Islet capture microplate in the XF Analyzer and start measurement.
  10. Data analysis is performed with the seahorse analysis software Wave.


    Figure 2. Microscopic image of epidermis in the XF24 islet capture microplate. The epidermis has to be applied as flat as possible with the basal side in contact with the capture screen and then placed in the multiwell plate using the capture screen insert tool. The measurement of oxygen consumption takes place over the surface within the ring marked with the arrow (A). Within the ring in the center of the net, there should be no holes (B) or air bubbles (D). Flat-mounted epidermis, without air bubbles, which covers the entire surface and with the right orientation (C). Schematic setup of the XF24 measuring system with an islet capture microplate (E) (Modified Image from Schniertshauer et al., 2018). Scale bars = 2 mm.

Programming the XF controller
This section describes the individual program steps of the XF controller for measuring the oxygen consumption rate as shown in Figure 2. The stress reagents are diluted to a final concentration of 4 μM with XF assay medium. The concentration of Oligomycin, FCCP and Rotenone/Antimycin A should be determined in a preliminary experiment for the respective experimental conditions. The injection of the stress reagent simultaneously should be carried out in all wells.
Workflow of the XF controller:

  1. Calibrate Probes
  2. Time of Delay: 15 min
  3. First Loop–3 times (Basal state)
    1. Mixing: 4 min
    2. Time of Delay: 2 min
    3. Measurement: 3 min
  4. Injection of Port A (50 μl Oligomycin, 4 μM final concentration)
    1. Use oligomycin to block the ATP synthase. 
    2. Then equivalent the decrease in OCR to the amount of oxygen used for the synthesis of ATP.
  5. Second Loop–6 times (decrease of ATP-linked respiration)
    1. Mixing: 4 min
    2. Time of Delay: 2 min
    3. Measurement: 3 min
  6. Injection of Port B (55 μl FCCP, 4 μM final concentration)
    The decoupling of the respiratory chain by FCCP allows the maximal respiration and the spare respiratory capacity to be determined independently of the proton gradient.
  7. Third Loop–3 times (potentially uncoupled respiration)
    1. Mixing: 4 min
    2. Time of Delay: 2 min
    3. Measurement: 3 min
  8. Injection of Port C (60 μl Rotenone/Antimycin A, 4 μM final concentration)
    In the last Loop, complexes I and II are blocked by the addition of Rotenone and Antimycin A. The remaining oxygen consumption thus depends only on the non-mitochondrial respiration.
  9. Fourth Loop–6 times (non-mitochondrial respiration)
    1. Mixing: 4 min
    2. Time of Delay: 2 min
    3. Measurement: 3 min
  10. End of Program

Data analysis

Data can be analyzed using Prism 7.04 (GraphPad Software, Inc.). Values are presented as mean ± SEM, or individual values. Five replicates per experimental group consisting of one biopsy are recommended.

Representative data
Measurement of oxygen consumption rate in epidermal tissue derived from human skin biopsies according to the proceeding steps shows the typical course after addition of the four stress reagents Oligomycin, FCCP, Rotenone/Antimycin A as depicted in Figure 3.


Figure 3. Oxygen consumption rate (OCR) in epithelial tissue derived from human epidermal biopsies. Scheme depicting the main stages after injecting the four active ingredients in three injecting steps. This makes it possible to determine the efficiency of the individual complexes within the respiratory chain (n = 5, mean ± SEM).

Notes

  1. The measurement of oxygen consumption rates in epithelial tissue derived from human epidermal biopsies we described here was highly reproducible.
  2. After surgery, biopsies should be kept in PBS at 4 °C for a maximum of 36 h. It is important to carry out the OCR measurement in this time frame as the respiratory parameters decrease significantly after this period.
  3. The concentration of Oligomycin, FCCP and Rotenone/Antimycin A should be determined in a preliminary experiment for the respective experimental conditions. The indicated concentrations may differ for other biopsies. In preliminary studies, the concentration of 4 μM was determined. Lower concentrations show, in contrast to monolayer cell cultures, no or little effects. 
  4. Because a biopsy is a cell complex in which the substances simply take longer to penetrate the entire sample, a higher concentration and more measurement point as stated in the Seahorse manual must be used.
  5. In order to exclude photoaging-effect, only biopsies obtained from the same body region should be compared. Samples from body regions with a high UV exposure (e.g., eyelids) show a significantly lower mitochondrial respiration on average than samples with a low UV exposure (e.g., abdomen) from donors with the same age.

Recipes

  1. 10 M NaOH
    40 g NaOH
    ad 100 ml ddH2O
  2. 1x PBS
    8 g NaCl
    0.20 g KCl
    2.88 g Na2HPO4·12H2O
    1.24 g KH2PO4
    ad 1 L ddH2O, pH 7.4
  3. Enzymatic digestion solution
    8 ml KBMTM Gold Keratinocyte Growth Medium BulletKitTM
    4 ml Dispase II
    Final concentration 2.4 U/ml
  4. XF assay medium
    0.675 g Dulbecco’s Modified Eagle’s Medium-high glucose
    ad 50 ml ddH2O, sterile filtering, pH 7.4

Acknowledgments

The authors thank Dr. Hug, Dr. Huth, Dr. Astfalk and Dr. Then-Schlagau for the kind supply of samples. They also thank Dr. Franz Enzmann and Dr. Alexander Bürkle for scientific advice. This work was supported with funds from the Baden-Württemberg Ministry of Science, Research and Art and the BMBF–FHprofUnt2012 “MitoFunk” [03FH022PX2].

Competing interests

One of the authors has consulting contracts with MSE Pharmazeutika GmbH, Bad Homburg, Germany and Beiersdorf AG, Hamburg, Germany.

Ethics

All experiments were conducted in accordance with the Declaration of Helsinki and approved by the Ethics Commission of the State Medical Association of Baden-Württemberg, Germany (187-03). Patients were informed in advance and gave their written consent to the use of their samples.

References

  1. Furda, A. M., Marrangoni, A. M., Lokshin, A. and Van Houten, B. (2012). Oxidants and not alkylating agents induce rapid mtDNA loss and mitochondrial dysfunction. DNA Repair (Amst) 11(8): 684-692.
  2. Gebhard, D., Matt, K., Burger, K. and Bergemann, J. (2014). Shortwave UV-induced damage as part of the solar damage spectrum is not a major contributor to mitochondrial dysfunction. J Biochem Mol Toxicol 28(6): 256-262.
  3. Harman, D. (1972). The biologic clock: the mitochondria? J Am Geriatr Soc 20(4): 145-147.
  4. Schniertshauer, D., Gebhard, D. and Bergemann, J. (2018). Age-dependent loss of mitochondrial function in epithelial tissue can be reversed by coenzyme Q10. J Aging Res 2018: 6354680.
  5. Yoshida, T., Goto, S., Kawakatsu, M., Urata, Y. and Li, T. S. (2012). Mitochondrial dysfunction, a probable cause of persistent oxidative stress after exposure to ionizing radiation. Free Radic Res 46(2): 147-153.

简介

皮肤细胞经常受到环境影响,如空气污染,化学物质,病原体和紫外线辐射。紫外线辐射可以破坏不同的生物结构,但最重要的是细胞DNA。线粒体含有自己的基因组,并在很大程度上积累紫外线诱导的DNA突变。这可能导致例如,加速皮肤老化。因此,了解有害外部影响对线粒体功能的影响对于更好地了解与年龄有关的疾病的发展至关重要。以前的研究是针对源自原代细胞的细胞培养物进行的,这些细胞培养物不能完全代表皮肤的真实情况,而线粒体参数几乎不考虑或根本不考虑。在这里,我们描述了一种使用Agilent Seahorse XF24 Flux Analyzer测量人皮肤活检组织上皮组织中线粒体呼吸参数的方法。在测定之前,表皮和真皮被酶促分离,然后我们使用XF24胰岛捕获微孔板定位表皮样品以测量氧消耗速率(OCR)和细胞外酸化速率(ECAR)。在这些板中,小网可以固定在板底部。表皮放置在网上的生命基底侧。连续注入三个端口中的活性成分以确定每种化合物的效果。这允许确定呼吸链内各个复合物的效率。该协议能够测试有毒物质及其对人上皮组织中线粒体呼吸参数的影响。
【背景】人体皮肤是人体最大的器官,可作为物理屏障,保护身体免受许多有害外部因素的影响,如空气污染或太阳辐射(Gebhard et al。,2014)。太阳辐射,例如,诱导活性氧(ROS)的产生增加,从而导致DNA损伤和皮肤突变,导致皮肤老化和皮肤癌(Gebhard 等。,2014)。由DNA损伤引起的线粒体功能障碍被认为在这些和其他必需的细胞过程中起重要作用。活性氧(ROS) - 损伤的线粒体DNA(mtDNA)和蛋白质的积累可以诱导电子传递链(ETC)内的线粒体功能障碍,进而导致ROS产生增加和线粒体功能障碍增加(Furda et al 。,2012; Yoshida et al。,2012)。根据“衰老的线粒体理论”,这种恶性循环是细胞衰老,组织功能障碍和退化的主要原因(Harman,1972)。因此,了解有害外部影响对线粒体功能的影响对于更好地了解老化一般是必不可少的。不幸的是,细胞培养中线粒体呼吸的测量不能完全反映皮肤的真实情况。因此,我们建立了一种有效的方法,使用Seahorse XF24 Flux Analyzer和Seahorse XF24胰岛捕获微量培养板直接测量人表皮活检中的线粒体呼吸离体。 XF24分析仪是一种多孔板系统,可通过固态荧光团荧光的变化来测量氧消耗率(OCR)和细胞外酸化率(ECAR)。在该系统中,可以使用多达四个不同的端口来注射化合物以获得线粒体呼吸谱。该方案中描述的程序包括活组织检查的制备,用于插入XF24分析仪的表皮的制备以及通过添加特定线粒体抑制剂来测量线粒体参数。该方案用于测试有毒物质及其对人上皮组织中线粒体呼吸参数的影响。此外,可以根据供体的年龄测试各种物质的年龄相关效应。来自皮肤活组织检查的表皮中线粒体呼吸的分析代表了对现有筛查的重要补充。

关键字:耗氧率, 线粒体呼吸, 表皮, 老化, 皮肤活检, 离体

材料和试剂

  1. 离心管15,50 ml(SARSTEDT,目录号:62.554.001,62.547.254)
  2. 显微镜载玻片(Carl Roth,目录号:1879.1)
  3. 培养皿(SARSTEDT,目录号:83.3902)
  4. 移液器吸头10μl,20μl,200μl,1,000μl(SARSTEDT,目录号:70.1130,70.116,70.760.002,70.762)
  5. 无菌过滤器0.2μm(SARSTEDT,目录号:83.1826.001)
  6. 人体皮肤活检(在4°C,1x PBS中保存最多36小时)
  7. 2-丙醇(Carl Roth,目录号:9866.1)
  8. Dispase II(中性蛋白酶,II级)(罗氏,目录号:04942078001,储存温度4°C)
  9. Dulbecco改良的Eagle's培养基,高葡萄糖(Sigma-Aldrich,目录号:D7777-10L,储存温度4°C)
  10. KBM TM 金角质形成细胞生长培养基BulletKit TM (Lonza,目录号:00192060,储存温度4°C)
  11. KCl(Carl Roth,目录号:6781.3)
  12. KH 2 PO 4 (Carl Roth,目录号:3904.2)
  13. Na 2 HPO 4 •12H 2 O(Carl Roth,目录号:N350.1)
  14. NaCl(Carl Roth,目录号:3957.3)
  15. NaOH(Carl Roth,目录号:6771.3)
  16. Seahorse XF Calibrant Solution(安捷伦科技,产品目录号:100840-000)
  17. Seahorse XF细胞Mito压力测试试剂盒(Oligomycin,FCCP,Rotenone / Antimycin A)(安捷伦科技,目录号:103015-100,储存温度-20°C)
  18. 10 M NaOH(参见食谱)
  19. 1x PBS(见食谱)
  20. 酶消化液(参见食谱)
  21. XF检测培养基(见食谱)

设备

  1. 剪刀,不锈钢(Carl Roth,目录号:HCX4.1)
  2. 海马捕获屏插入工具(安捷伦科技,目录号:101135-100)
  3. Seahorse XF24胰岛捕获FluxPak含有传感器盒和胰岛捕获微孔板(Agilent Technologies,目录号:101174-100)
  4. 镊子,不锈钢(Carl Roth,目录号:2687.1)
  5. 37°C孵化器,无CO 2 (GFL,目录号:4010)
  6. pH计FiveEasy TM F20(Mettler Toledo,目录号:30266626)
  7. 移液器,Eppendorf研究®加10μl,20μl,200μl,1,000μl(Eppendorf,目录号:3123000020,3123000039,3123000055,3123000063)
  8. Seahorse XF24 Flux Analyzer(安捷伦科技,型号:XF24,目录号:100737-100)

软件

  1. Seahorse XF24 Flux Analyzer软件(仪器软件)(Agilent Technologies,Version 1.8.1.1)
  2. Wave(分析软件)(Agilent Technologies,Version 2.6)

程序

分析前一天

  1. 从样品管中取活组织检查,先用70%异丙醇洗涤,然后用1x PBS洗涤,除去残留的血液和其他杂质。
  2. 在下一步中,去除脂肪和组织(见图1A和1B)。
  3. 将活组织切片(见图1B)切成小块(约0.4×0.4cm)(见图1C),并将它们置于15ml离心管中的酶消化溶液中。
  4. 将活组织检查储存于4°C酶消化16小时(在非搅拌条件下)。 
  5. 在非CO 2 培养箱中测量前一天根据制造商的说明在Seahorse XF Calibrant中对传感器盒进行水合。
  6. 打开XF24分析仪,将系统预热至37°C。


    图1.活组织检查的准备步骤。 :一种。粘附于真皮/表皮的脂肪和结缔组织(箭头)。 B.第一次纯化步骤后的真皮/表皮。 C.用于酶消化的溶液中的分开的活组织检查。 D.与Dispase II孵育后分离的表皮片。 E.使用胰岛捕获屏幕捕获筛选插入工具(Agilent Technologies)。

分析日
  1. 准备XF测定培养基并加热至37°C 
  2. 在酶消化后,小心地从真皮(镊子)移除表皮并将其置于具有1x PBS的培养皿中(参见图1D)。在该步骤中,重要的是确保基底面向下朝向培养皿的底部。表皮片应放置在捕获屏幕上的生命基底侧。这种方向对于测量至关重要,因为传感器会检测到面向捕获屏幕的一侧(参见图2E)。 
  3. 用显微镜载玻片小心地从1x PBS中提起表皮,然后在第二张幻灯片上擦拭表皮(翻过表皮一次)。
  4. 用捕获屏幕上的第二张幻灯片擦去皮肤,将其平滑地拉出,然后将捕获屏幕插入工具插入胰岛捕获微孔板(参见图1E)。表皮应覆盖捕获屏幕的整个表面,以确保始终测量相同的表皮区域(参见图2A-2C)。表皮下应该没有气泡(见图2D),因为它们会干扰测量。
  5. 用镊子去除井中多余的悬垂皮肤。
  6. 将450μlXF测定培养基添加到胰岛捕获微板的每个孔中。
  7. 将胰岛捕获微孔板在37℃孵育孵育器(不含CO 2 )45分钟,以将表皮调节至新培养基。
  8. 按照编程加载传感器盒的进样口(参见XF控制器的编程),按照XF分析仪的说明开始校准传感器盒。
  9. 校准完成后,将胰岛捕获微孔板放入XF分析仪中并开始测量。
  10. 使用海马分析软件Wave进行数据分析。


    图2. XF24胰岛捕获微孔板中表皮的显微图像。表皮必须尽可能平坦,基部侧与捕获屏接触,然后放入多孔板中使用捕获屏幕插入工具。氧气消耗的测量发生在标有箭头(A)的环内的表面上。在网中心的环内,不应有孔(B)或气泡(D)。平面安装的表皮,没有气泡,覆盖整个表面并具有正确的方向(C)。具有胰岛捕获微孔板(E)的XF24测量系统的示意性设置(来自Schniertshauer的改良图像等人,,2018)。比例尺= 2毫米。

对XF控制器进行编程
本节描述了用于测量氧消耗速率的XF控制器的各个程序步骤,如图2所示。使用XF测定培养基将应激试剂稀释至终浓度为4μM。 Oligomycin,FCCP和鱼藤酮/抗霉素A的浓度应在各实验条件的初步实验中确定。应同时注入应力试剂应在所有孔中进行。
XF控制器的工作流程:

  1. 校准探针
  2. 延迟时间:15分钟
  3. 第一循环-3次(基础状态)
    1. 混合:4分钟
    2. 延迟时间:2分钟
    3. 测量:3分钟
  4. 注射端口A(50μl寡霉素,4μM终浓度)
    1. 使用寡霉素阻断ATP合成酶。 
    2. 然后将OCR降低到用于合成ATP的氧气量。
  5. 第二次循环-6次(ATP连锁呼吸减少)
    1. 混合:4分钟
    2. 延迟时间:2分钟
    3. 测量:3分钟
  6. 注射端口B(55μlFCCP,4μM终浓度)
    通过FCCP解耦呼吸链允许独立于质子梯度确定最大呼吸和备用呼吸能力。
  7. 第三次循环-3次(潜在的解耦呼吸)
    1. 混合:4分钟
    2. 延迟时间:2分钟
    3. 测量:3分钟
  8. 注射端口C(60μl鱼藤酮/抗霉素A,终浓度4μM)
    在最后一个循环中,通过添加鱼藤酮和抗霉素A来阻断复合物I和II。因此剩余的氧消耗仅取决于非线粒体呼吸。
  9. 第四次循环-6次(非线粒体呼吸)
    1. 混合:4分钟
    2. 延迟时间:2分钟
    3. 测量:3分钟
  10. 计划结束

数据分析

可以使用Prism 7.04(GraphPad Software,Inc。)分析数据。值表示为平均值±SEM或单个值。建议每个实验组重复5次,包括一次活组织检查。

代表性数据
根据前述步骤测量来自人皮肤活组织检查的表皮组织中的氧消耗速率,显示加入四种应激试剂Oligomycin,FCCP,Rotenone / Antimycin A后的典型过程,如图3所示。


图3.源自人表皮活组织检查的上皮组织中的耗氧率(OCR)。描述在三个注射步骤中注射四种活性成分后的主要阶段的方案。这使得可以确定呼吸链内各个复合物的效率(n = 5,平均值±SEM)。

笔记

  1. 我们在此描述的来自人表皮活组织检查的上皮组织中的氧消耗率的测量具有高度可重复性。
  2. 手术后,活组织检查应在4°C的PBS中保存最多36小时。在此时间段内进行OCR测量非常重要,因为呼吸参数在此期间后会明显减少。
  3. Oligomycin,FCCP和鱼藤酮/抗霉素A的浓度应在各实验条件的初步实验中确定。其他活组织检查的指示浓度可能不同。在初步研究中,确定了4μM的浓度。与单层细胞培养物相比,较低的浓度显示没有或几乎没有效果。 
  4. 因为活组织检查是一种细胞复合物,其中物质需要更长的时间才能穿透整个样本,因此必须使用海马手册中规定的更高浓度和更多测量点。
  5. 为了排除光老化效应,应仅比较从相同身体区域获得的活组织检查。来自身体区域的具有高紫外线照射(例如,眼睑)的样本显示出比来自供体的低UV暴露(例如,腹部)的样本平均线粒体呼吸显着降低与年龄相同。

食谱

  1. 10 M NaOH
    40克NaOH
    ad 100 ml ddH 2 O.
  2. 1x PBS
    8克NaCl
    0.20克KCl
    2.88 g Na 2 HPO 4 •12H 2 O
    1.24克KH 2 PO 4
    ad 1 L ddH 2 O,pH 7.4
  3. 酶消化解决方案
    8 ml KBM TM 金角质形成细胞生长培养基BulletKit TM
    4毫升Dispase II
    最终浓度2.4 U / ml
  4. XF检测培养基
    0.675克Dulbecco's改良Eagle的中高葡萄糖
    ad 50 ml ddH 2 O,无菌过滤,pH 7.4

致谢

作者感谢Hug博士,Huth博士,Astfalk博士和Then-Schlagau博士提供的样品供应。他们还感谢Franz Enzmann博士和AlexanderBürkle博士的科学建议。这项工作得到了巴登 - 符腾堡州科学,研究和艺术部以及BMBF-FHprofUnt2012“MitoFunk”[03FH022PX2]的资助。

利益争夺

其中一位作者与德国Bad Homburg的MSE Pharmazeutika GmbH和德国汉堡的Beiersdorf AG签订了咨询合同。

伦理

所有实验均按照赫尔辛基宣言进行,并经德国巴登 - 符腾堡州医学会伦理委员会批准(187-03)。事先通知患者,并书面同意使用他们的样本。

参考

  1. Furda,A。M.,Marrangoni,A。M.,Lokshin,A。和Van Houten,B。(2012)。 氧化剂而非烷化剂会导致mtDNA快速丢失和线粒体功能障碍。 DNA修理(Amst) 11(8):684-692。
  2. Gebhard,D.,Matt,K.,Burger,K。和Bergemann,J。(2014)。 作为太阳损伤谱的一部分,短波紫外线引起的损伤不是线粒体功能障碍的主要原因。 J Biochem Mol Toxicol 28(6):256-262。
  3. Harman,D。(1972)。 生物钟:线粒体? J Am Geriatr Soc 20(4):145-147。
  4. Schniertshauer,D.,Gebhard,D。和Bergemann,J。(2018)。 辅酶Q10可以逆转上皮组织中年龄依赖性的线粒体功能丧失。 J Aging Res 2018:6354680。
  5. Yoshida,T.,Goto,S.,Kawakatsu,M.,Urata,Y。和Li,T。S.(2012)。 线粒体功能障碍,是接触电离辐射后持续氧化应激的可能原因。 Free Radic Res 46(2):147-153。
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引用:Schniertshauer, D., Gebhard, D. and Bergemann, J. (2019). A New Efficient Method for Measuring Oxygen Consumption Rate Directly ex vivo in Human Epidermal Biopsies. Bio-protocol 9(5): e3185. DOI: 10.21769/BioProtoc.3185.
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