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EdU labeling of Trypanosome Cells and Their Kinetoplast DNA (kDNA)
EdU标记锥体虫细胞及其动质体DNA(kDNA)   

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Abstract

Trypanosome mitochondrial genome, known as Kinetoplast DNA (kDNA), is a massive network of interlocked DNA rings. The studies of kDNA replication and architecture are of major significance since kDNA is a valid drug target. However, DNA in procyclic trypanosomes can not be labeled with tracer concentrations of 3[H]-thymidine, possibly because they lack a high-affinity transporter for thymidine. Therefore, BrdU, a thymidine analog, has been used at high concentrations to study kDNA replication. However, the detection of BrdU with anti-BrdU antibody requires harsh conditions such as the acid or heat treatment to seperate double DNA strand, which affects the ability for other antibodies to bind as well as the morphology and ability for dyes that require dsDNA to bind efficiently. Instead, EdU (5-Ethynyl-2′-deoxyuridine), a novel thymidine analog, can be used to study kDNA replication and cell proliferation with a simplified protocol. Detection of EdU is based on a click reaction, which is a copper (I) catalyzed reaction between an azide and an alkyne. This click reaction does not require DNA denaturation and it is multiplex compatible, such as other antibodies and dyes for cell cycle analyses. To visualize trypanosome replicating nuclear DNA and kDNA, EdU is added into the medium of cell culture and incubated for 0.5 h to 3 h and then detected by the following procedures.

Keywords: Trypanosome(锥虫), Kinetoplast DNA(动基体的DNA), EdU labeling(埃杜标记), KDNA(kDNA), Trypanosoma brucei(布氏锥虫)

Materials and Reagents

  1. Click-iT® Cell Reaction Buffer Kit (Life Technologies, InvitrogenTM, catalog number: C10269 )
  2. EdU (Life Technologies, InvitrogenTM, catalog number: A10044 )
  3. Alexa Fluor® 488 azide (Life Technologies, InvitrogenTM, catalog number: A10266 )
    OR Alexa Fluor® 594 azide (Life Technologies, InvitrogenTM, catalog number: C10270 )
  4. 4% Paraformaldehyde (PFA) in PBS
  5. Deionized water (ddH2O)
  6. Poly-L-Lysine (Sigma-Aldrich, catalog number: P8920 )
  7. TEFLON printed slides 8-well, 6 mm diameter (Electron Microscopy Sciences)
  8. Vectashield® mounting medium with DAPI (Vector Laboratories, catalog number: H-1200 )
  9. Nail polish
  10. EdU solution (molecular weight of EdU: 252.22 g/mol) (see Recipes)
  11. 0.1 M Glycine in 1x PBS (see Recipes)

Equipment

  1. A humid chamber (a box with moistened paper tower; can protect from light and at least 8 x 10 cm2)
  2. Microscope Coverslips, 22 x 50 cm2 (Fisherbrand®)
  3. Centrifuge
  4. 28 °C 5% CO2 Cell culture incubator
  5. Fluorescent microscope 

Procedure


I.   EdU labeling of trypanosome cells

Notes:
1) This protocol has not been used for the bloodstream form of trypanosomes yet. However, it is supposed to work well for the bloodstream form of trypanosomes too.
2) From step 3 to the end of the protocol, operate at room temperature (RT).
3) From step 9, please protect from light by covering the box with lid or the eppendorf tubes with foil paper. In steps 7, 8, and 10, use a 100 or 200 μl pipette to remove solution from each well.

  1. Culture procyclic trypanosomes in 10 ml SDM-79 medium supplemented with 10% FBS at 28 °C, 5% CO2 incubator.
  2. Add EdU solution to cell culture at final concentration of 50 μM to 100 μM and continue to incubate at the same condition as step 1 for 30 min to 3 h as planned.
  3. Trypanosome cells are then pelleted at 700 x g for 5 min.
  4. Wash the cells with 1 ml 1x PBS once and resuspend cells in 1x PBS at around 2 x 107 cells/ml.
  5. Coat the 8-well glass slide with poly-L-lysine (0.1% w/v in H2O).
  6. Add 25 μl cell suspension onto each well in a humid chamber (see Equipment 1) and seat for 5 min.
  7. Remove cell suspension and add 40 μl 4% paraformaldehyde to fix cells for 5 min.
  8. Remove fixatives and wash cells twice with 50 μl 0.1 M Glycine for 5 min.
  9. Prepare Click-iT reaction cocktail (make cocktail fresh and keep dark).
    For 200 μl (1 slide or 8 wells, 25 μl/well):
    Component A: 1x TBS                   176 μl
    Component B: 100 mM CuSO4      4 μl
    Component C: Buffer additive        20 μl
    1 mM Alexa-488-azide                   1 μl
    (OR 1 mM Alexa-594-azide            1 μl)
  10. Remove the wash buffer from each well at step 8 and add 25 μl reaction cocktail per well and incubated for 1 h at RT. Protect from light.
  11. After 1 h incubation, remove the reaction cocktail, wash twice with 50 μl 1x PBS. Protect from light.
  12. Mount slides with Vectashield Mounting Medium with DAPI (1.5 μg/ml), seal with nail polish and seat for 15~30 min. Protect from light.
  13. Examine by Fluorescent Microscopy (using 63x or 100x objective lens) (Figure 1).


    Figure 1. EdU labeling of procyclic form of trypanosome cells. DNA synthesis was measured by adding 100 μM EdU to the culture medium for 1 h before harvest. Cells were then adhered to a poly-L-lysine coated 8-well slide and EdU was detected with Alexa-488-azide followed by the counterstaining of DNA and nucleus with 1.5 μg/ml DAPI. The inset in the EdU panel is the enlarged EdU labeling image of the cell on the right in the Phase panel. N, nucleus; k, kDNA. Red, DAPI; Green, EdU. Bar, 5 μm.

II.  EdU labeling of kDNA networks isolated from trypanosomes

Notes:
1) From step 3 to the end of the protocol, operate at RT (room temperature).
2) From step 7, please protect from light by covering the box with lid or the eppendorf tubes with foil paper.
3) In steps 8 and 9, please use a 100 or 200 μl pipette to remove solution from each well.

  1. Culture the procyclic trypanosomes in SDM-79 medium supplemented with 10% FBS at 28 °C, 5% CO2 incubator. For kDNA isolation, prepare more than 1 x 108 cells in total.
  2. Add EdU solution to cell culture at 50 μM to 100 μM and continue to incubate for 30 min to 3 h as planned.
  3. Isolate kDNA networks from ≥ 5 x 107 trypanosome cells.
  4. Coat the 8-well glass slide with 1:50 diluted poly-L-lysine in ddH2O.
  5. Mix 1 μl isolated kDNA networks with 19 μl 1x PBS (add 1x PBS in each well first and then mix kDNA with 1x PBS on the well).
    Note: Before doing EdU labeling of isolated kDNA networks, it is suggested to examine kDNA networks abundance by DAPI staining only by following steps 5, 6, 10 and 11 (at least 15 kDNA networks in each field to continue).
  6. Allow kDNA networks seat on the slide (poly-L-lysine coated) for 30 min at room temperature in humid chamber (see Equipment 1).
  7. Prepare Click-iT reaction cocktail (make cocktail fresh and keep dark).
    For 200 μl (1 slide or 8 wells, 25 μl/well):
    Component A: 1x TBS                 176 μl
    Component B: 100 mM CuSO4    4 μl
    Component C: Buffer additive      20 μl
    1 mM Alexa-488-azide                 1 μl
    (OR 1 mM Alexa-594-azide          1 μl)
  8. Remove kDNA and 1x PBS mixture from each well and add 25 μl reaction cocktail per well and incubated for 1 h at RT. Protect from light.
  9. After 1 h incubation, remove the reaction cocktail, wash twice with 50 μl 1x PBS. Protect from light.
  10. Mount slides with Vectashield Mounting Medium with DAPI (1.5 μg/ml), and seal with nail polish, and seat for 15~30 min. Protect from light.
  11. Examine by Fluorescent Microscopy (using 100x objective lens) (Figure 2).


    Figure 2. EdU labeling of isolated kDNA network from trypanosome cells. Networks were isolated from EdU-labeled cells and adhered to an 8-well glass slide and incorporated EdU was then detected with Alexa-488-azide (in green) and networks were stained with 1.5 μg/ml DAPI (in red). Arrows, a unit-sized pre-replication kDNA; Arrowhead, a double-sized post-replication kDNA.

Recipes

  1. EdU solution (molecular weight of EdU: 252.22 g/mol)
    Dissolve 12.16 mg EdU in 1 ml ddH2O for 50 mM EdU solution
    Dissolve 12.16 mg EdU in 1 ml ddH2O for 50 mM EdU solution
  2. 1x PBS
    Dissolve the following in 800 ml distilled H2O
    8 g of NaCl
    0.2 g of KCl
    1.44 g of Na2HPO4
    0.24 g of KH2PO4
    Adjust pH to 7.4; then adjust volume to 1 L with additional distilled H2O. Sterilize by autoclaving.

Acknowledgments

I thank all lab members of Paul Englund and Robert Jensen for helpful discussions. This work was supported by NIH grant AI058613.

References

  1. Wang, J., Englund, P. T. and Jensen, R. E. (2012). TbPIF8, a Trypanosoma brucei protein related to the yeast Pif1 helicase, is essential for cell viability and mitochondrial genome maintenance. Mol Microbiol 83(3): 471-485.

简介

锥虫体线粒体基因组,称为Kinetoplast DNA(kDNA),是互锁DNA环的大块网络。 kDNA复制和结构的研究具有重要意义,因为kDNA是有效的药物靶标。然而,在循环锥虫中的DNA不能用示踪剂浓度的3 H] - 胸苷标记,可能是因为它们缺少用于胸苷的高亲和性转运蛋白。因此,已经以高浓度使用BrdU(胸苷类似物)来研究kDNA复制。然而,用抗BrdU抗体检测BrdU需要苛刻的条件,例如分离双DNA链的酸或热处理,其影响其它抗体结合的能力以及需要dsDNA结合的染料的形态和能力有效率的。相反,EdU(5-乙炔基-2'-脱氧尿苷),一种新的胸苷类似物,可以用于研究kDNA复制和细胞增殖与简化的协议。 EdU的检测基于点击反应,其是叠氮化物和炔之间的铜(I)催化反应。这种点击反应不需要DNA变性,并且是多重兼容的,例如用于细胞周期分析的其他抗体和染料。为了显现锥虫复制的核DNA和kDNA,将EdU加入细胞培养基的培养基中,孵育0.5小时至3小时,然后通过以下程序检测。

关键字:锥虫, 动基体的DNA, 埃杜标记, kDNA, 布氏锥虫

材料和试剂

  1. Click-iT细胞反应缓冲液试剂盒(Life Technologies,Invitrogen TM ,目录号:C10269)
  2. EdU(Life Technologies,Invitrogen TM,目录号:A10044)
  3. Alexa Fluor 488叠氮化物(Life Technologies,Invitrogen TM ,目录号:A10266)
    或Alexa Fluor 594叠氮化物(Life Technologies,Invitrogen TM ,目录号:C10270)
  4. 4%多聚甲醛(PFA)在PBS中的溶液
  5. 去离子水(ddH 2 O)
  6. 聚-L-赖氨酸(Sigma-Aldrich,目录号:P8920)
  7. TEFLON印刷载片8孔,直径6mm(Electron Microscopy Sciences)
  8. 使用DAPI(Vector Laboratories,目录号:H-1200)的Vectashield
  9. 指甲油
  10. EdU溶液(EdU的分子量:252.22g/mol)(参见配方)
  11. 0.1 M甘氨酸(1 x PBS)(参见配方)

设备

  1. 湿室(具有润湿的纸塔的箱子;可以防止光和至少8×10cm <2>)
  2. 显微镜盖玻片,22×50cm 2 (Fisherbrand )
  3. 离心机
  4. 28℃5%CO 2细胞培养箱
  5. 荧光显微镜

程序


I.  锥体虫细胞的EdU标记

注意:
1)该方案尚未用于锥体的血流形式。 但是,它也应该对锥体的血液形式也很好。
2)从步骤3到协议结束,在室温( RT )下操作。 3)从第9步,通过用盖子或埃彭多夫管用箔纸覆盖盒子避免光照。 在步骤7,8和10中,使用100或200μl移液管从每个孔中除去溶液。

  1. 在28℃,5%CO 2培养箱中,在补充有10%FBS的10ml SDM-79培养基中培养原环状锥虫。
  2. 向细胞培养物中加入EdU溶液,最终浓度为50μM至100μM,并继续按照计划在与步骤1相同的条件下孵育30分钟至3小时。
  3. 然后将锥虫体细胞在700×g下沉淀5分钟
  4. 用1ml 1×PBS洗涤细胞一次,并在1×PBS中以约2×10 7个细胞/ml重悬细胞。
  5. 用聚-L-赖氨酸(0.1%w/v H 2 O)涂覆8孔玻璃载玻片。
  6. 添加25微升细胞悬液在潮湿的房间(见设备1)的每个孔,座位5分钟
  7. 取出细胞悬液,加入40μl4%多聚甲醛固定细胞5分钟
  8. 取出固定液,用50μl0.1 M甘氨酸洗涤细胞两次,每次5分钟
  9. 准备Click-iT反应鸡尾酒(使鸡尾酒新鲜,保持黑暗) 对于200μl(1个载玻片或8个孔,25μl/孔):
    组件A:1x TBS                 176μl
    组分B:100mM CuSO 4。     4微升
    组分C:缓冲添加剂&      20微升
    1mM Alexa-488-叠氮化物                1微升
    (或1微升Alexa-594叠氮化物       1微升)
  10. 在步骤8从每个孔中删除洗涤缓冲液,每孔加入25μl反应混合液,并在室温下孵育1小时。防光。
  11. 孵育1小时后,取出反应混合液,用50μl1×PBS洗涤两次。防光。
  12. 安装幻灯片与Vectashield安装介质与DAPI(1.5微克/毫升),密封指甲油和座位15〜30分钟。防光。
  13. 通过荧光显微镜检查(使用63x或100x物镜)(图1)

    图1.原核细胞形式的锥体虫细胞的EdU标记。通过在收获前向培养基中加入100μMEdU 1小时来测量DNA合成。然后将细胞粘附到聚-L-赖氨酸包被的8孔载玻片上,用Alexa-488-叠氮化物检测EdU,随后用1.5μg/ml DAPI对DNA和核进行复染。 EdU面板中的插图是相位面板右侧的单元的放大的EdU标记图像。 N,核; k,kDNA。红色,DAPI; Green,EdU。条,5μm。

II。  EdU标记从锥虫分离的kDNA网络

注意:
1)从步骤3到协议结束,在RT(室温)下操作。
2)从第7步,请用盖子或埃彭多夫管用铝箔纸覆盖盒子避免光照。
3)在步骤8和9中,请使用100或200μl移液管从每个孔中除去溶液。

  1. 在补充有10%FBS的SDM-79培养基中在28℃,5%CO 2培养箱中培养原环形锥体虫。 对于kDNA分离,总共制备超过1×10 8个细胞
  2. 以50μM至100μM添加EdU溶液至细胞培养物,并继续按计划孵育30分钟至3小时。
  3. 分离≥5×10 7个锥体虫体细胞的kDNA网络。
  4. 将8孔玻璃载玻片与1:50稀释的聚-D-赖氨酸在ddH 2 O中涂覆。
  5. 将1μl分离的kDNA网络与19μl1×PBS(首先在每个孔中加入1×PBS,然后将kDNA与1×PBS在孔上混合)混合。
    注意:在对分离的kDNA网络进行EdU标记之前,建议仅通过以下步骤5,6,10和11(每个区域中至少15个kDNA网络继续)通过DAPI染色检查kDNA网络丰度。
  6. 允许kDNA网络在玻片上(聚-L-赖氨酸涂层)在潮湿室中室温下30分钟(参见设备1)。
  7. 准备Click-iT反应鸡尾酒(使鸡尾酒新鲜,保持黑暗) 对于200μl(1个载玻片或8个孔,25μl/孔):
    组件A:1x TBS               176μl
    组分B:100mM CuSO 4。   4微升
    组分C:缓冲添加剂&    20微升
    1mM Alexa-488-叠氮化物              1微升
    (或1微升Alexa-594叠氮化物       1微升)
  8. 从每个孔中删除kDNA和1×PBS混合物,并添加25μl反应混合物每孔,并在室温下孵育1小时。防光。
  9. 孵育1小时后,取出反应混合液,用50μl1×PBS洗涤两次。防光。
  10. 用Vectashield固定介质用DAPI(1.5μg/ml)装载载玻片,并用指甲油密封,并座15-30分钟。防光。
  11. 通过荧光显微镜检查(使用100倍物镜)(图2)

    图2.来自锥体虫细胞的分离的kDNA网络的EdU标记。从EdU标记的细胞分离网络,并粘附到8孔玻璃载玻片上,然后加入Alexa-488-叠氮化物 绿色),网络用1.5μg/ml DAPI(红色)染色。 箭头,单位大小的复制前kDNA; 箭头,双倍大小的复制后kDNA

食谱

  1. EdU溶液(EdU的分子量:252.22g/mol) 将12.16mg EdU溶解在1ml ddH 2 O中用于50mM EdU溶液
    将12.16mg EdU溶解在1ml ddH 2 O中用于50mM EdU溶液
  2. 1x PBS
    将以下物质溶于800ml蒸馏H 2 O中 8克NaCl
    0.2克KCl
    1.44g的Na 2 HPO 4
    0.24g的KH 2 PO 4 sub/
    调节pH至7.4; 然后用另外的蒸馏H 2 O调节体积至1L。 通过高压灭菌消毒。

致谢

我感谢Paul Englund和Robert Jensen的所有实验室成员进行了有益的讨论。 这项工作是由NIH授权AI058613支持。

参考文献

  1. Wang,J.,Englund,P.T.and Jensen,R.E。(2012)。 TbPIF8,与酵母Pif1解旋酶相关的锥虫蛋白 对细胞活力和线粒体基因组维持至关重要。 Mol Microbiol 83(3):471-485。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, J. (2013). EdU labeling of Trypanosome Cells and Their Kinetoplast DNA (kDNA). Bio-protocol 3(12): e799. DOI: 10.21769/BioProtoc.799.
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