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[Bio101] Cell Cycle Analysis and GFP Expression of Zebrafish Embryos by FACS (Originally by Narie Storer)
[Bio101] 通过流式细胞仪研究斑马鱼胚胎中细胞周期分析和绿色荧光蛋白的表达   

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Abstract

Cell cycle analysis in combination of GFP expression has been well used to study the cell cycle distribution of GFP labeled cells. This protocol is a method to analyze the cell cycles of GFP marked cells from zebrafish embryos.

Materials and Reagents

  1. Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 14040 )
  2. Fetal bovine serum (FBS)
  3. Ethanol
  4. PFA (USB)
  5. EtOH
  6. Formaldehyde
  7. RNaseA
  8. Hoechst 33342 solution (Life Technologies, Molecular Probes, catalog number: H3570 )
  9. 40 μm cell strainer (BD Biosciences, catalog number: 352340 )
  10. Polystyrene FACS tube (BD Biosciences, Falcon®, catalog number: 352054 )
  11. 50 ml Falcon tube (BD Biosciences, catalog number: 352070 )
  12. PI solution (see Recipes)
  13. PBS + 5% FBS (see Recipes)

Equipment

  1. FACS
  2. Standard tabletop centrifuges  
  3. Water bath
  4. Polystyrene FACS tube
  5. Cell strainer

Procedure

  1. Prepare embryos for FACS
    1. Thaw FBS in 37 °C bath.
    2. Make PBS + 5% FBS.
    3. Place embryos in a 5 ml polystyrene FACS tube (50 embryos per analysis).
    4. Suck off as much liquid as possible and add 1 ml PBS/FBS.
    5. Homogenize embryos at ~1/4 speed maximum for a few sec. 
    6. Check to see that there are no clumps of embryo/cells.
    7. Label 1x 50 ml tubes for each tube of embryos.
    8. Put 40 μm cell strainer onto 1 set of 50 ml tubes.
    9. Pipet homogenized embryos onto filter.
    10. Rinse tube with 1 ml PBS/FBS and pipet onto filter.
    11. Transfer filtered embryos into a 5 ml polypropylene FACS tube.
    12. Centrifuge at 2,000 rpm, 5 min.
    13. Pipet off supernatant.
      If not fixing, resuspend in 500 μl PBS/FBS and leave on ice.

  2. Fix cells with formaldehyde and fix/permeabilize with ethanol
    1. Resuspend cells gently in 500 μl cold 0.9x PBS.
    2. Add 500 μl cold 2% PFA (in 0.9x PBS). Mix gently.
    3. Incubate for 30 min, on ice (make sure 70% EtOH in freezer).
    4. Centrifuge at 2,000 rpm, 5 min, at 4 °C.
    5. Pipet off supernatant (collect in separate PFA waste container) and wash once in 1 ml cold 0.9x PBS.
    6. Centrifuge at 2,000 rpm, 5 min, at 4 °C.
    7. Pipet off supernatant.
      For Hoechst staining, go directly from PFA fix to Hoechst protocol below.
    8. Slowly add 1 ml 70% ethanol at -20 °C drop wise to the cell pellet with the tube sitting on a vortex. 
    9. Incubate for 30 min, on ice. 
    10. While incubating, prepare PI solution (In foil).
    11. Centrifuge at 2,000 rpm, 5 min, at 4 °C.
    12. Resuspend in 500 μl PI solution.
      For cells that are just being fixed, without PI, resuspend in 500 μl 0.9x PBS.
    13. Incubate at room temperature ~30 min, in dark.
    14. Filter cells on 40 μm filter into 50 ml tube.
    15. Transfer cells to 5 ml polystyrene FACS tube.
    16. Place on ice then bring to FACS.

  3. Hoechst staining
    1. Prepare Hoechst 33342 solution—1:1,000 of 1 mg/ml stock in 0.9x PBS.
    2. Incubate at 28 °C for ~30 min, in dark.
    3. Filter cells on 40 μm filter into 50 ml tube.
    4. Transfer cells to 5 ml polystyrene FACS tube.
    5. Place on ice then bring to the FACS equipment.

Recipes

  1. PI solution (in foil)
    PI + RNaseA in 0.9x PBS
    500 μl 1 mg/ml PI stock (20x)
    100 μl 10 mg/ml RNase A stock (1 μl/ml)
    9.4 ml 0.9x PBS
  2. PBS + 5% FBS
    9.5 ml 0.9x PBS + 0.5 ml FBS

Acknowledgments

This protocol was modified from the original protocol created and developed by Narie Storer in the Len Zon lab at Boston Children’s Hospital, Boston, USA and this work was supported by NIH grant R01 HL04880-21.

简介

细胞周期分析与GFP表达的组合已经被良好地用于研究GFP标记的细胞的细胞周期分布。 这个协议是一种方法来分析GFP标记的细胞从斑马鱼胚胎的细胞周期

材料和试剂

  1. 磷酸盐缓冲盐水(PBS)(Life Technologies,Invitrogen TM,目录号:14040)
  2. 胎牛血清(FBS)
  3. 乙醇
  4. PFA(USB)
  5. EtOH
  6. 甲醛
  7. RNaseA
  8. Hoechst 33342溶液(Life Technologies,Molecular Probes,目录号:H3570)
  9. 40μm细胞过滤器(BD Biosciences,目录号:352340)
  10. 聚苯乙烯FACS管(BD Biosciences,Falcon ,目录号:352054)
  11. 50ml Falcon管(BD Biosciences,目录号:352070)
  12. PI解决方案(参见配方)
  13. PBS + 5%FBS(参见配方)

设备

  1. FACS
  2. 标准台式离心机&
  3. 水浴
  4. 聚苯乙烯FACS管
  5. 细胞过滤器

程序

  1. 准备胚胎的FACS
    1. 解冻FBS在37℃浴。
    2. 制作PBS + 5%FBS。
    3. 将胚胎放置在5ml聚苯乙烯FACS管(每个分析50个胚胎)。
    4. 抽出尽可能多的液体,加入1ml PBS/FBS。
    5. 以〜1/4速度最大值匀浆胚胎几秒钟。
    6. 检查看到没有胚胎/细胞团块。
    7. 标签1x 50毫升管的每个胚胎管。
    8. 将40微米的细胞过滤器置于1套50毫升管中。
    9. 吸管将胚胎匀浆至过滤器上。
    10. 用1ml PBS/FBS冲洗管,吸取到过滤器上。
    11. 转移过滤的胚胎到5ml聚丙烯FACS管。
    12. 以2,000rpm离心5分钟。
    13. 吸出上清液。
      如果不固定,重悬在500μlPBS/FBS中,并留在冰上
  2. 用甲醛固定细胞并用乙醇固定/透化
    1. 重悬细胞轻轻在500微升冷0.9x PBS。
    2. 加入500μl冷的2%PFA(在0.9x PBS中)。 轻轻混合。
    3. 在冰上孵育30分钟(确保在冰箱中70%EtOH)。
    4. 在4℃下以2,000rpm离心5分钟。
    5. 吸出上清液(收集在单独的PFA废物容器中),并在1ml冷的0.9x PBS中洗涤一次。
    6. 在4℃下以2,000rpm离心5分钟。
    7. 吸出上清液。
      对于Hoechst染色,直接从PFA修正到Hoechst方案如下。
    8. 在-20℃下将1ml 70%乙醇缓慢加入到细胞沉淀中,将管置于涡旋上。
    9. 在冰上孵育30分钟。
    10. 孵育时,准备PI溶液(In foil)。
    11. 在4℃下以2,000rpm离心5分钟。
    12. 重悬于500μlPI溶液中 对于刚刚固定的细胞,无PI,重悬于500μl0.9x PBS中。
    13. 在室温下孵育〜30分钟,在黑暗中。
    14. 将40μm过滤器上的细胞过滤到50ml管中。
    15. 转移细胞到5ml聚苯乙烯FACS管。
    16. 放在冰上然后送到FACS
  3. Hoechst染色
    1. 制备Hoechst 33342溶液-1:1,000μL在0.9x PBS中的1mg/ml储备液。
    2. 在28℃下在黑暗中孵育〜30分钟。
    3. 将40μm过滤器上的细胞过滤到50ml管中。
    4. 转移细胞到5ml聚苯乙烯FACS管。
    5. 放在冰上,然后带到FACS设备

食谱

  1. PI溶液(箔)
    PI + RNaseA在0.9x PBS中的溶液 500μl1mg/ml PI原液(20x)
    100μl10mg/ml RNA酶A储液(1μl/ml) 9.4ml 0.9x PBS
  2. PBS + 5%FBS
    9.5ml 0.9x PBS + 0.5ml FBS

致谢

该协议是由Narie Storer在Len Zon实验室在波士顿儿童医院,波士顿,美国创建和开发的原始协议修改的,这项工作由NIH授权R01 HL04880-21支持。

  • English
  • 中文翻译
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jing, L. (2012). Cell Cycle Analysis and GFP Expression of Zebrafish Embryos by FACS (Originally by Narie Storer). Bio-protocol Bio101: e185. DOI: 10.21769/BioProtoc.185;
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9/19/2012 11:36:48 PM Reply
Lili Jing
Department of Cell and Molecular Biology, University of Pennsylvania, USA

可以用homogenizer打碎,或者用Pellet pestle手工研碎。
Pellet Pestle 可以从sigma购买。

9/24/2012 9:36:50 PM


9/17/2012 10:12:08 PM Reply
Lili Jing
Department of Cell and Molecular Biology, University of Pennsylvania, USA

Hi,

Sorry that there was a mistake in the Chinese translation. All "PBS/FB" should be "PBS/FBS" instead. Thanks for pointing this out.

PBS/FBS is

PBS + 5%FBS:9.5ml 0.9x PBS + 0.5ml FBS

9/18/2012 6:37:13 PM