Molecular Techniques and Methods

Flow Cytometric Analysis of Isolated Nuclei from Plant Tissue

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

Tris Buffer (500 ml)
0.01 M Tris-HCl (pH 7.5) ------------------------- 5 ml of 1 M Tris-HCl
0.01 M Na2-EDTA ------------------------------- 10 ml of 0.5 M Na2-EDTA
0.1 M NaCl --------------------------------------- 10 ml of 5 M NaCl
Distilled H2O -------------------------------------- 475 ml


Formaldehyde Fixative (100 ml)
0.01 M Tris-HCl (pH 7.5) ------------------------ 1 ml of 1 M Tris-HCl
0.01 M Na2-EDTA ------------------------------- 2 ml of 0.5 M Na2-EDTA
0.1 M NaCl --------------------------------------- 2 ml of 5 M NaCl
Triton X-100 -------------------------------------- 250 ul
2-4% Formaldehyde ------------------------------- 5.4-11.2 ml of 37% Formaldehyde
Distilled H2O --------------------------------------- 89 ml
  • Prepare just before use!


    Lysis Buffer (200 ml)
    0.015 M Tris-HCl (pH 7.5) ------------------------ 3 ml of 1 M Tris-HCl
    0.002 M Na2-EDTA ------------------------------- 0.8 ml of 0.5 M Na2-EDTA
    0.08 M KCl ---------------------------------------- 16 ml of 1 M KCl
    Spermine tetrahydrochloride ------------------------ 34.8 mg
    0.1 M NaCl ---------------------------------------- 5 ml of 5 M NaCl
    Triton X-100 --------------------------------------- 250 ul
    2-Mercaptoethanol --------------------------------- 220 ul
    Add deionized water to make a final volume of -----200 ml
  • Filter through a 0.22 um filter.
  • Store at -20oC.


    DAPI Stock Solution (0.1 mg/ml)
    DAPI ---------------------------------------------- 5 mg
    Deionized H2O ------------------------------------ 50 ml
  • Dissolve by stirring for 60 min.
  • Filter 0.22 um filter and store at -20oC.




    PROCEDURES

    1. Harvest the root tips (1 cm) into deionized H2O.

    2. Fix roots in a Formaldehyde Fixative at 4oC for 20-30 min.

    3. Wash the roots in Tris Buffer for 5 min at 4oC.
  • Repeat 3 times.

    4. Excise root meristems (1-2 mm) and transfer into Dounce homogenizer containing 1 ml of Lysis Buffer.

    5. Isolate the nuclei by homogenizing 10-20 sec.

    6. Filter the suspension through a 50 um nylon mesh.
  • Nuclei is about 10 um in size.

    7. Stain the nuclei by adding DAPI Stock Solution to final concentration of 2 ug/ml.

    8. Align flow cytometer with suitable calibration particles.

    9. Run nuclei isolated from control tissues and adjust the gain of the instrument so that the G1 peak appears on a suitable channel.

    10. Analyze the fluorescence intensity of DAPI-stained nuclei isolated from samples.




    NOTES




    KIT INFORMATION




    REFERENCES

  • Dolezel, J, Cihalikova, J, Weiserova, J, Lucretti, S (1999) Cell cycle synchronization in plant root meristems. Methods in Cell Science 21: 95-107.

  • Grogan, MW, Collins, JM (1990) In "Guide to flow cytometry methods." Marcel Dekker, Inc. New York and Basel.



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