FGRS: protocol

 

Cell Synchrony

Last Updated

June 28, 2010 6:11 AM


Much of this information comes from:

Cell cycle checkpoint controls by Howard B. Lieberman, 2004


Hydroxyurea & General Protocol


Hydroxyurea blocks cells in S phase by inhibiting the production of dNTPs.  Cells visually arrest with medium sized buds.


The following is the protocol we will use for hydroxyurea cell cycle block-and-release:


    Initial Cell Growth

  1. 1.Grow a 5 mL overnight culture to saturation.

  2. 2.Inoculate a 35 mL culture to a target OD of 0.3 (between OD 0.2 and 0.4) from the 5 mL culture in the morning.

   
    Drug Treatment

  1. 3.Add hydroxyurea to a final culture concentration of 0.2M.

    We have made lab stock (4°C) of hydroxyurea in a 50 mL conical tube.  Per the instructions on the tube, use 5 mL of the stock for a 35 mL culture.  Please be sure to vortex the stock of hydroxyurea before use - it often comes out of solution in storage.  This type of thing should be considered for any chilled solution of this nature.

  2. 4.Allow the cells to grow for an additional 1.5 hours after drug treatment.

   
    Establish Synchrony (Block)

  1. 5.Use microscopy to examine the cell culture.

    Cells that enter but do not complete S phase will bud (small buds) but not divide.  You are looking and waiting until the culture is ~95% in this state before declaring the culture to be synchronized.  Examine the cells every 30 minutes until this is true.

    Note: See the figure below for an example of S phase (Monitoring Methods).
    Note: Take 3 µl of cells for each monitoring time-point - be sure to leave the culture in the incubator as much as possible to allow the cells to progress to block step.


    Remove Drug Treatment (Release)

  1. 6.Spin cells down completely (normal procedure) and wash the cells twice with pre-warmed YPD of the same volume (~40 mL).  Fully re-suspend cells with gentle pipetting for each wash.  Spin cells down completely between each wash.

  2. 7.After the second wash and spin-down re-suspend the cells in 40 mL of this pre-warmed YPD.

    You must remember to have 120 mL of YPD pre-warmed medium ready for this step in order to not shock the cells and thereby affect the experiment in progress.


    Monitoring & Experimentation

  1. 8.Begin time-course experimentation.

    You should also consider monitoring the synchronization of cells using one of the monitoring methods detailed below.  Note that the time-course experimentation will vary from situation to situation.  Refer to other protocols for exact steps.


Nocodazole


Nocodazole blocks microtubule polymerization and holds cells in G2/M.  Cells will have large buds and twice the DNA content due to the fact that they have completed S phase.


The concentration of nocodazole in the final cell culture is 15 µg / mL.  The stock in the 4°C is designed for you to use 2.5 mL of stock per 35 mL culture volume.


The methods for nocodazole use are exactly the same as for hydroxyurea.  Arrested cells will have much larger buds that are nearly the same size as the daughter cell.  Please see the protocol above and follow it closely.


Alpha Factor


Alpha factor works with MATa yeast cells and blocks them at the G1/S boundary by inhibiting Cln-Cdc28 activity.  Cells arrest in G1 with a definitive “schmoo” morphology.


Cells recover rapidly from alpha factor treatment and progress through two or three cell cycles synchronized.


Hormone Stock Concentration

The stock in the -20°C is concentrated at 2.5 µg / µL.
(in PKILLION box, by itself at the moment)


Alpha Factor Primary Treatment

The initial concentration of alpha factor in the cell culture is intended to be 5.0 µg / mL.

  1. STEP:
    You will add 2 µL of the alpha factor stock for every 1 mL of culture.


Like nocodazole, the methods for alpha factor use are exactly the same as for hydroxyurea.  Please see the protocol above and follow it closely.  There is one difference, however - secondary treatment.


Alpha Factor Secondary Treatment

One hour after the primary treatment you will add an additional secondary treatment of 5.0 µg / mL to the growing culture.  This is important and should be recorded for your experiments (make a note on any growth curves where both primary and secondary treatment occur).

  1. STEP:
    You will add an additional 2 µL of the alpha factor stock for every 1 mL of culture.


Wait an additional hour (after this second treatment).


Your culture should now be synchronized and can/should be visualized.


What does schmoo look like (see Figure B)?




α-Pheromone-Induced “Schmooing” and Gene Regulation Require White-Opaque Switching during Candida albicans Mating, Shawn R. Lockhart, Rui Zhao, Karla J. Daniels, and David R. Soll*


Summary:


  1. 1.Start overnight.

  2. 2.Next day, start culture @ OD ~ 0.3.

  3. 3.Add alpha factor (2 uL per 1 mL of culture).

  4. 4.Wait an hour.

  5. 5.Add alpha factor (2 uL per 1 mL of culture).

  6. 6.Wait an hour.

  7. 7.Cells should now be synchronized.

  8. 8.Visualize with microscope.

  9. 9.If synchronized, you can release (use YPD media to wash).

  10. 10.Re-suspend in the volume of media you were using.

  11. 11.Experiment starts - get time points every X minutes.



Monitoring Methods


Percentage Budding

Use the microscopy methods discussed above during Establish Synchrony to monitor percentages of budded cells at the next entry of S phase after release.  Note that this method requires you to monitor cells every 30 minutes.


Though you will be visualizing only 15 µl it is recommended that you take 500 µl from the cell culture at each time point.


Important Note: You *must* sonicate the 500 µl before visualization in order to ensure separation of cells.  Use 50% power, 15 seconds.  The tip of the sonicator should be fairly deep in the microcentrifuge tube so as to prevent spilling or spraying of the culture.


This sonication must be done in order to completely separate the cells for analysis.  You will want to count total cells per a 4x4 matrix and schmoo cells.  Use these numbers to establish a percent schmoo.





Image from University of Sheffield


Gene Expression

We can use gene expression to monitor and track cell cycle progression.  Additionally, gene expression can be used to establish time-points post-drug-release for the individual stages of the cell cycle.


The following genes will be used as markers for specific stages of the cell cycle:


  1. 1.CLN2 - G1/S Boundary

  2. 2.H2A - S Phase

  3. 3.CLB4 - G2

  4. 4.CLB2 - M Phase

  5. 5.EGT2 - M/G1 Boundary


We will be doing RTPCR (reverse transcription PCR) to quantitatively compare the transcriptional abundance of these marker genes across a time-course of a synchronized culture.