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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.

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[Bio101] Cell Cycle Analysis and GFP Expression of Zebrafish Embryos by FACS (Originally by Narie Storer)

Cell Biology > Cell imaging > Fluorescence
Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
2/20/2012, 8745 views, 2 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.185

[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.



How to cite: 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; Full Text



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9/19/2012 11:36:48 PM  

是怎样让斑马鱼从组织消化成细胞的?难道仅靠震荡就可以

9/24/2012 9:36:50 PM  

Lili Jing (Author)
Department of Cell and Molecular Biology,University of Pennsylvania

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

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9/17/2012 10:12:08 PM  

FB是什么?
并添加1ml PBS / FB。

9/18/2012 6:37:13 PM  

Lili Jing (Author)
Department of Cell and Molecular Biology,University of Pennsylvania

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

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Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
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