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Quantification of Cell Corpses, Cell Death Occurrence, Cell Corpse Duration
死细胞、细胞死亡概率以及死细胞持续时间的量化分析   

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Abstract

During the development of the C. elegans hermaphrodite, 131 of the 1090 somatic cells generated undergo programmed cell death, among which 113 die during embryogenesis starting from 200-cell stage. The apoptotic cells (also called “cell corpses”) appear as highly refractile button-like objects and are easily identified using differential interference contrast (DIC) optics (Robertson et al., 1982).

Materials and Reagents

  1. Agar pad (made by melting and coating 4% agar on glass slides)
  2. C. elegans strains [Wild type (N2), Engulfment-defective mutants: commonly used mutants including ced-1, ced-5, ced-7, ced-6, ced-2, ced-12, ced-10, which all contain persistent cell corpses that are easily detected under DIC optics]
  3. KH2PO4
  4. Na2HPO4
  5. NaCl
  6. MgSO4
  7. Peptone
  8. Cholesterol
  9. Vaseline
  10. NGM agar (see Recipes)
  11. M9 (used to mount embryos on agar pads) (see Recipes)

Equipment

  1. Zeiss Axioimager M1 microscope (Carl Zeiss)
  2. AxioCam monochrome digital camera (Carl Zeiss)
  3. AxiovisionRel 4.7 software (Carl Zeiss)
  4. Slides
  5. Coverslips

Procedure

  1. Quantification of cell corpses
    C. elegans is grown on NGM agar plates carrying a lawn of bacterial and kept at 20 °C. Embryos are randomly picked from NGM agar plates containing mix-staged worms and mounted on slides with agar pads in M9 and covered with coverslips. Cell corpses are observed using a Zeiss Axioimager M1 microscope equipped with DIC at 20 °C. Cell corpses are identified by the “raised button” morphology and quantified in the head region of living embryos either at the six different embryonic stages (comma, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold) for a time course analysis (Figure 2) or at the 4-fold embryonic stage. 15 embryos are counted at each embryonic stage for each strain.


    Figure 1. Seven Apoptotic cells (arrows) which appear as “raised button” objects in a C. elegans embryo are shown. Some apoptotic cells are not visible at this focus plane.

    Figure 2. Time-course analysis of cell corpses during embryonic development was performed in wild-type (N2, open bar), nrf-5(qx16) (black bar), or nrf-5(sa513) (gray bar). At least 15 embryos were scored at each stage. Data are shown as mean+SEM. Data derived from N2 and nrf-5(qx16) or N2 and nrf-5(sa513) were compared by unpaired t test. **P < 0.0001; all other points had P > 0.05.

  2. Monitor the occurrence of embryonic cell death and cell corpse duration
    C. elegans embryos at the two-cell stage are mounted on slides with agar pads in M9 and coverslips are sealed with Vaseline. Images in a 20 micron z series (0.5 micron per section) were captured every 1 min for 8 h using a Zeiss Axioimager M1 microscope equipped with an AxioCam monochrome digital camera (Carl Zeiss). Images are processed and viewed using AxiovisionRel 4.7 software (Carl Zeiss). Embryonic cell deaths are followed during 200-370 min after the first embryonic cleavage by the appearance of the “raised button” morphology of cell corpses. The duration of cell corpses is determined by following the appearance and disappearance (no button-like morphology can be seen) of apoptotic cells. At least three embryos from each strain are followed and quantified. The standard error of the mean (SEM) is used as y error bars for bar charts plotted from the mean value of the data. Data derived from different genetic backgrounds were compared by Student’s two way unpaired t-test. Data were considered statistically different at P < 0.05.

Recipes

  1. M9
    0.022 M KH2PO4
    0.042 M Na2HPO4
    0.086 M NaCl
    0.001 M MgSO4
  2. NGM agar
    NaCl 3 g
    Agar 17 g
    Peptone 2.5 g
    Cholesterol (5 mg/ml in EtOH) 1 ml
    H2O 975 ml

Acknowledgments

This protocol is adapted from Robertson et al. (1982) and Stanfield and Horvitz (2000).

References

  1. Robertson, A. M. G. and Thomson, J. N. (1982). Morphology of programmed cell death in the ventral nerve cord of Caenorhabditis elegans larvae. J Embryol Exper Morphol 67(1): 89-100.
  2. Stanfield, G. M. and Horvitz, H. R. (2000). The ced-8 gene controls the timing of programmed cell deaths in C. elegans. Mol Cell 5(3): 423-433.

简介

在C的开发期间。 elegans 雌雄同体,所产生的1090个体细胞中的131个经历程序性细胞死亡,其中113个在从200个细胞阶段开始的胚胎发生期间死亡。 凋亡细胞(也称为"细胞尸体")表现为高折射的纽扣状物体,并且使用微分干涉对比(DIC)光学器件(Robertson等人,1982)容易地鉴定。

材料和试剂

  1. 琼脂垫(通过熔化并在载玻片上涂覆4%琼脂制成)
  2. C。 线虫菌株[野生型(N2),Engulfment缺陷突变体:通常使用的突变体包括ced-1,ced-5,ced-7,ced-6,ced-2,ced-12,ced- 它们都包含在DIC光学下容易检测到的持续性细胞尸体]
  3. KH 2 PO 4
  4. Na HPO 4
  5. NaCl
  6. MgSO 4 4 /
  7. 蛋白胨
  8. 胆固醇
  9. 凡士林
  10. NGM琼脂(见配方)
  11. M9(用于在胚胎垫上安装胚胎)(参见配方)

设备

  1. Zeiss Axioimager M1显微镜(Carl Zeiss)
  2. AxioCam单色数码相机(Carl Zeiss)
  3. AxiovisionRel 4.7软件(Carl Zeiss)
  4. 幻灯片
  5. 盖舌

程序

  1. 细胞尸体的定量
    C。 elegans在含有细菌细菌的NGM琼脂平板上生长并保持在20℃。从含有混合阶段蠕虫的NGM琼脂平板上随机挑取胚胎,并且将其安装在具有M9中的琼脂垫的载玻片上并用盖玻片覆盖。使用装备有DIC的Zeiss Axioimager M1显微镜在20℃下观察细胞尸体。通过"升高的按钮"形态鉴定细胞尸体,并在活的胚胎的头部区域中在六个不同的胚胎阶段(逗号,1.5倍,2倍,2.5倍,3倍,4倍)对于时间过程分析(图2)或在4倍胚胎阶段。在每个胚胎阶段对每个菌株计数15个胚胎

    图1 七个凋亡细胞(箭头)在 C中显示为"凸起按钮"对象。线虫 胚胎。一些凋亡细胞在这个焦平面是不可见的。

    图2。 时间过程  在胚胎发育期间进行细胞尸体的分析 野生型(N2,空心柱),nrf-5(qx16)(黑色柱)或nrf-5(sa513)(灰色 bar)。每个阶段至少有15个胚胎得分。数据 显示为平均值±SEM。从N2和nrf-5(qx16)或N2和 nrf-5(sa513)进行比较。 ** P < 0.0001;所有 其他点具有P> 0.05。

  2. 监测胚胎细胞死亡和细胞尸体持续时间的发生率
    C。 elegans胚胎在两细胞阶段安装在具有琼脂垫的载玻片上,M9中,盖玻片用凡士林密封。使用配备有AxioCam单色数字照相机(Carl Zeiss)的Zeiss Axioimager M1显微镜,每1分钟捕获20微米z系列(每个截面0.5微米)的图像8小时。使用AxiovisionRel 4.7软件(Carl Zeiss)处理和观察图像。在第一次胚胎裂解后的200-370分钟期间,通过细胞尸体的"升高的按钮"形态的出现跟踪胚胎细胞死亡。细胞尸体的持续时间通过跟踪凋亡细胞的出现和消失(没有看到按钮样形态)来确定。来自每个菌株的至少三个胚胎被跟踪并定量。平均值的标准误差(SEM)用作从数据的平均值绘制的条形图的y误差条。通过学生双向非配对t检验比较来自不同遗传背景的数据。认为数据在P < 0.05。

食谱

  1. M9
    0.022 M KH 2 PO 4 sub/
    0.042M Na 2 HPO 4
    0.086M NaCl
    0.001M MgSO 4
  2. NGM琼脂
    NaCl 3 g
    琼脂17克
    蛋白胨2.5 g
    胆固醇(5mg/ml,在EtOH中)1ml
    H O 975ml

致谢

该协议改编自Robertson等人(1982)和Stanfield和Horvitz(2000)。

参考文献

  1. Robertson,A.M.G。和Thomson,J.N。(1982)。 Caenorhabditis elegans的腹侧神经索中程序性细胞死亡的形态学幼虫。 Embryol Exper Morphol 67(1):89-100。
  2. Stanfield,G.M。和Horvitz,H.R。(2000)。 ced-8基因控制em中程序性细胞死亡的时间。 elegans 。 Mol Cell 5(3):423-433。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, Y., Wang, H. and Wang, X. (2013). Quantification of Cell Corpses, Cell Death Occurrence, Cell Corpse Duration. Bio-protocol 3(9): e693. DOI: 10.21769/BioProtoc.693.
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Awani Awani
Pennstate university
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