Ex vivo Analysis of DNA Repair Capacity of Human Peripheral Blood Mononuclear Cells by a Modified Host Cell Reactivation Assay
利用改进的宿主细胞再活化反应进行人外周血单核细胞的DNA修复能力的离体检测
发布: 2019年08月05日第9卷第15期 DOI: 10.21769/BioProtoc.3325 浏览次数: 4240
评审: Lu SongAnonymous reviewer(s)
PDF 
Q&A
参见作者原研究论文
The authors used this protocol in:
Mar 2016
Abstract
The ability of humans to repair DNA damages decreases with increasing age. In order to be able to repair daily occurring DNA damages, it becomes more and more important to preserve repair capability of cells with aging. The preservation of DNA repair processes contributes to preventing DNA mutations and subsequently the onset of age-related diseases such as cancer. For the determination of DNA repair of human cells, mostly in vitro cell cultures are used. However, an ex vivo approach can provide a more accurate result compared with in vitro cell cultures, since the DNA repair ability is measured directly without the influence of prolonged culture time. Published protocols use in vitro cultured cells with a single reporter plasmid or a luciferase reporter. Our modified host cell reactivation assay enables the measurement of DNA repair capacity (nucleotide excision repair) of ex vivo isolated human peripheral blood mononuclear cells (PBMCs). For this purpose, PBMCs are isolated out of human anticoagulated blood by density gradient centrifugation. Directly after isolation, the PBMCs are co-transfected with two plasmids, one being previously damaged by UVC irradiation and one remaining undamaged. PBMCs are incubated for 24 h and subsequently analyzed by fluorescence activated cell sorting (FACS). The ability of cells to repair the DNA damages leads to a functional reactivation of the reporter gene. The assay presented here provides a solution to determine human DNA repair capacity ex vivo directly out of the human body. Furthermore, it can be used to research the ex vivo influence of different substances on DNA repair capacity of humans.
Keywords: Modified host cell reactivation assay (改进的宿主细胞再活化反应)
DNA repair capacity (DNA修复能力)
Human peripheral blood mononuclear cells (人外周血单核细胞)
Ex vivo (离体)
Nucleotide excision repair (核苷酸剪切修复)
Aging (衰老)
Background
Maintaining the integrity of the human genome is a prerequisite for delaying or preventing the development of age-related diseases. Every day, up to 50,000 DNA-damaging events occur in every cell (reviewed in Shrinivas et al., 2017). Such damages arise because of exogenous influences like radiation or chemicals, or via the normal cellular metabolism. For this reason, functional DNA repair mechanisms are crucial for the survival of organisms. One of the most important DNA repair pathways is nucleotide excision repair (NER), which detects and eliminates bulky DNA lesions and helix-distorting DNA adducts like cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts (6-4PPs) (for review see Nouspikel, 2009).
The modified host cell reactivation assay (mHCRA) is a reliable and reproducible method for the research of NER capacity of human cells based on the restoration of a previously damaged reporter gene. The principle of reporter gene restoration was first described in 1985 (Protić-Sabljić et al., 1985), subsequently adjusted by Athas et al. for an application with human lymphocytes (Athas et al., 1991) and further refined by using fluorescent reporter proteins (Roguev and Russev, 2000). Up to now, few researchers use the mHCRA, although it is a highly reproducible and reliable method. Qiao et al. used the modified HCRA with a luciferase reporter on human lymphocytes to study DNA repair (Qiao et al., 2002). In 2010, Burger et al. published an advanced method, which describes the mHCRA for studies on human skin cells. They used two reporter plasmids and fluorescence-activated cell sorting to determine DNA repair capacity (Burger et al., 2010). Furthermore, Mendez et al. researched DNA repair by carrying out nucleofection of cryopreserved human lymphocytes with one reporter plasmid (Mendez et al., 2011).
Our mHCRA enables the measurement of NER capacity of PBMCs as an ex vivo approach. By using two different plasmids, transfection efficiencies can be normalized by a transfected control plasmid. This procedure allows for comparing experiments with different transfection efficiencies with each other. We used the method described here to reveal the influence of calorie reduction on the DNA repair capacity of humans (Matt et al., 2016). Moreover, the use of this assay can provide a more accurate assessment of positive or negative effects of substances like dietary supplements or pharmaceutical agents on DNA repair ability of human cells than the use of in vitro cell cultures.
Materials and Reagents
- Centrifuge tubes 15, 50 ml (SARSTEDT, catalog numbers: 62.554.001, 62.547.254)
- 1.5 ml reaction tube (SARSTEDT, catalog number: 72.706)
- Petri dishes (SARSTEDT, catalog number: 83.3902)
- Pipette tips 10 μl, 20 μl, 200 μl, 1,000 μl (SARSTEDT, catalog numbers: 70.1130, 70.116, 70.760.002, 70.762)
- Serological pipettes 5 ml and 10 ml (SARSTEDT, catalog numbers: 86.1253.001 and 86.1254.001)
- LeucosepTM, with porous barrier, 50 ml, pre filled with LeucosepTM separation medium (Greiner Bio-One GmbH, catalog number: 227288)
- Sterile filter 0.2 μm (SARSTEDT, catalog number: 83.1826.001)
- Cell culture plate, 6-well, Cell+ (SARSTEDT, catalog number: 83.3920.300)
- 0.4 cm electroporation cuvettes (Molecular BioProducts, Inc., purchased via Thermo Fisher Scientific GmbH, catalog number: 5540-11)
- FACS tubes 5 ml and 0.5 ml (SARSTEDT, catalog numbers: 55.1579, 55.673)
- Plasmid-DNA solution pEGFP-N1 (Clontech, catalog number: 6085-1) and pDsRedExpress-N1 (Clontech, catalog number: 632429), each 900 ng/µl in Qiagen EB buffer (storage temperature for longer periods: -80 °C, otherwise keep at 4 °C)
- Human venous blood, anticoagulated
- KCl (Carl Roth, catalog number: 6781.3)
- KH2PO4 (Carl Roth, catalog number: 3904.2)
- Na2HPO4·12H2O (Carl Roth, catalog number: N350.1)
- NaCl (Carl Roth, catalog number: 3957.3)
- S-Monovette® K3 EDTA 4.9 ml and 7.5 ml (SARSTEDT, catalog numbers: 04.1931.001 and 01.1605.001)
- RPMI 1640 without Phenol Red (Gibco® by Life Technologies, purchased via Fisher Scientific GmbH, catalog number: 11835-063, storage temp. 4 °C)
- Fetal Bovine Serum (Gibco® by Life Technologies, chased via Fisher Scientific GmbH, catalog number: 10270-106)
- EB Buffer, part of the QIAGEN Plasmid Plus Giga Kit (Qiagen GmbH, Hilden, catalog number: 12991)
- BD FACS Sheath Solution with Surfactant (BD Biosciences, catalog number: 336911)
- 1x PBS (see Recipes)
- 0.9% NaCl solution (see Recipes)
- PBMC medium (see Recipes)
Equipment
- Pipettes, Eppendorf Research® Plus 10 μl, 20 μl, 200 μl, 1,000 μl (Eppendorf, catalog numbers: 3123000020, 3123000039, 3123000055, 3123000063)
- Stratalinker® UV Crosslinker (Stratagene, catalog number: 400072, model: Model 1800)
- Centrifuge with swinging bucket rotor (Eppendorf, catalog numbers: 5805000010, 5804709004)
- Neubauer counting chamber improved (Carl Roth, catalog number: PC72.1)
- Gene Pulser II with Capacitance extender plus and Pulse controller plus (Bio-Rad Laboratories, Inc., catalog numbers: 165-2106, 165-2108, 165-2110)
- CO2 Incubator Hera cell 150 (Kendro Laboratory Products, catalog number: 51013568)
- BD FACSCalibur Flow Cytometry System with 488 nm laser (Becton, Dickinson and Company)
- -80 °C freezer HFU600TV (Thermo Fisher ScientificTM, catalog number: 11670823)
Software
- CellQuest Pro (Becton, Dickinson and Company, version 4.0.2)
- Prism (GraphPad Software, Inc.)
Procedure
文章信息
版权信息
© 2019 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Matt, K. C. and Bergemann, J. (2019). Ex vivo Analysis of DNA Repair Capacity of Human Peripheral Blood Mononuclear Cells by a Modified Host Cell Reactivation Assay. Bio-protocol 9(15): e3325. DOI: 10.21769/BioProtoc.3325.
Download Citation in RIS Format
分类
分子生物学 > DNA > DNA 损伤和修复
您对这篇实验方法有问题吗？
在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。
提问指南
+ 问题描述
写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。
发布问题
