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Analysis of Cell Cycle Synchrony

Microscopic Analysis of Cell Cycle Synchrony

This protocol is used to estimate the extent of mitotic synchrony and the frequency of cells in metaphase in synchronized root tips.

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

  1. Fix them in 3:1 fixative overnight at 4°C.
  2. Remove fixative with several washes in 70% ethanol.
  3. If needed, store the tips in 70% ethanol at 4°C.
  4. Wash the tips in several changes of deionized H2O.
  5. Hydrolyse tips in 5N HCl at room temperature for 25 min.
  6. Wash in deionized H2O and incubate in Schiff's reagent for 1 hour.
  7. Wash tips in deionized H2O and macerate for about 1 min in 45% (v/v) acetic acid.
  8. Cut off the darkly stained meristem tip and squash it in a drop of fructose syrup under an 18 x 18-mm coverslip.
  9. Prepare five different slides. On each slide, analyse at least 1000 cells and determine the proportion of cells in metaphase.

Notes

Squash preparations prepared in fructose syrup can be maintained for few days in a refrigerator but they are not permanent. To make permanent slides, perform the following procedure:

  1. Squash meristem tip in a drop of 45% (v/v) acetic acid.
  2. Immediately after that, place the slide on a block of dry ice.
  3. After freezing, peel off the coverslip.
  4. Dehydrate the slide in two changes of 96% ethanol in Coplin jars.
  5. Leave to air-dry overnight.
  6. Dip the slide in xylene and mount it in a drop of DePeX (Serva).

Flow Cytometric Analysis of Cell Cycle Synchrony

This procedure is used to estimate of cell cycle synchrony induced by hydroxyurea and APM treatments. The protocol is based on the analysis of DNA content of isolated nuclei after staining with 4-,6-diamidino-2-phenylindole (DAPI). This dye is excited at UV and thus the flow cytometer must be equipped with a light source providing excitation in UV. We use BD FACSVantage equipped with a Coherent Innova 305 argon-ion laser.

  1. Harvest the root tips (1 cm) in deionized H2O.
  2. Immediately transfer the root tips into 25 ml of formaldehyde fixative and fix them at 5°C for 30 min (legumes) or 20 min (cereals).
  3. Wash the roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them in 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer.
  5. Isolate nuclei by homogenising at 9500 rpm for 15 sec.
  6. Stain the nuclei in suspension by adding DAPI stock solution to final concentration of 2 µg/ml.
  7. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  8. Make sure that the flow cytometer is aligned well for univariate analysis and that the band-pass filter 424/44 is placed in front of the fluorescence 1 (FL1) detector.
  9. Introduce the sample; let it stabilise at appropriate flow rate (e.g., 200 particles/s).
  10. Set a gating region on a dot plot of forward scatter height (FSC-H) and FL1 pulse height (FL1-H) to exclude small debris and large clumps.
  11. Adjust photomultiplier voltage and amplification gains so that the peaks corresponding to G1 and G2 nuclei are seen on a histogram of FL1 pulse area (FL1-A).
  12. Analyse 10 - 20 thousand nuclei and save the result on a disc.

Notes

Although the nuclei suspension can be stored overnight, it is recommended to perform the analysis on the same day of isolation.