Cell cycle

Using ImageJ to Measure Cell Fluorescence

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Image J can be downloaded for free from here .
This guide can also be downloaded as a complete PDF here: Measuring Cell Fluorescence using ImageJ

Here is a very simple guide for determining the level of  fluorescence in a given region (e.g nucleus)

  1. Select the cell of interest using any of the drawing/selection tools (i.e. rectangle, circle, polygon or freeform)
  2. From the Analyze menu select “set measurements”. Make sure you have AREA, INTEGRATED DENSITY and MEAN GRAY VALUE selected (the rest can be ignored).
  3. Now select “Measure” from the analyze menu or hit cmd+m (apple). You should now see a popup box with a stack of values for that first cell.
  4. Now go and select a region next to your cell that has no fluroence, this will be your background.
    NB: the size is not important. If you want to be super accurate here take 3+ selections from around the cell.
  5. Repeat this step for the other cells in the field of view that you want to measure.
  6. Once you have finished, select all the data in the Results window, and copy (cmd+c) and paste (cmd+v) into a new excel worksheet (or similar program)
  7. Use this formula to calculate the corrected total cell fluorescence (CTCF).
    NB: You can use excel to perform this calculation for you.
    CTCF = Integrated Density – (Area of selected cell  X Mean fluorescence of background readings)

     
  8. Make a graph and your done. Notice that in this example that the rounded up mitotic cell appears to have a much higher level of staining, but this is actually due to its smaller size, which concentrates the staining in a smaller space. So if you just used the raw integrated density you would have data suggesting that the flattened cell has less staining then the rounded up one, when in reality they have a similar level of fluorescence.

How to Cite this if you wold like to:

We have used this method in these papers:

McCloy, R. A., Rogers, S., Caldon, C. E., Lorca, T., Castro, A., and Burgess, A. (2014) Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events. Cell Cycle 13, 1400–1412 [Link]

Burgess A, Vigneron S, Brioudes E, Labbé J-C, Lorca T & Castro A (2010) Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc Natl Acad Sci USA 107: 12564–12569

But you can also find a similar method published here:

Gavet O & Pines J (2010) Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis. Dev Cell 18: 533-543

And here:

Potapova TA, Sivakumar S, Flynn JN, Li R & Gorbsky GJ (2011) Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited. Mol Biol Cell 22: 1191–1206

And my apologies to any others that I have not mentioned.

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Congratulation to Sam our PhD student on his 1st Author Publication!

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Great news, we recently published a collaborative paper in the journal Cell Division with the Lab of Dr Liz Caldon here at the Garvan.
The title of the paper is “Cyclin E2 is the predominant E-cyclin associated with NPAT in breast cancer cells”, and you can find it online here

It also marked the inaugural 1st Author publication for our PhD student Sam!

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Public Talk “Killing Cancer One Cell at a Time ” now on YouTube

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Here is a recent talk I gave to some members of the public at the Garvan Institute of Medical Research.

It is a very general and simple over-view of explaining 1) how cells in your body proliferate, 2) how this goes wrong in cancer, 3) the challenges we are facing in treating and killing cancer, and 4) most importantly how we hoping to improve current treatments in the near future.

A big thanks to all the fantastic Garvan Foundation Team who hosted, filmed, and edited the event.

Immunofluorescence Guide

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The Mitchison Lab has an excellent guide on staining and fixing cells for Actin and Microtubules which is worth reading [Link]

Coverslips

Most coverslips come with a fine film coating to stop them sticking to each other. This can reduce the ability of coating agents such as poly-L lysine from working properly, and can thus reduce the ability of cells to properly adhear to the glass. As most mitotic cells ’round’ up and have a much weaker attachment, a poorly coated coverslip can dramatically reduce the numbers of cells you finally have to look at down the microscope. Thus it is always important to first clean the coverslips and then coat them with either Histogrip, Fibronectin, or Poly-L-lysine.

Cleaning Coverslips
1) Boil coverslips in dH2O in a large beaker for several minutes in a microwave
2) Add HCl to a final concentration of about 1M to the hot water. Careful of fumes do in a    fume hood if possible.
3) Cover the beaker with some parafilm, and gently stir/rock the coverslips on for 4-16h or until cool.
4) Rinse the coverslips several times in dH2O.
5) Then rinse 3-5x with 100% Ethanol, leave coverslips in EtOH and go to TC hood
6) In TC hood, separate individual coverslips out onto large piece of clean Kimwipe or similar blotting paper and allow to air dry.
7) They can now be stored (for unto a year) in a 10cm plate or 50ml falcon until coating. Some people like to autoclave them but it is not necessary.

Coating Coverslips
Histogrip (Invitrogen)
1) In a TC hood, make a 1/10 dilution of the histogrip into 100% Acetone in a 50ml Falcon tube. Normally 10-15ml final volume is plenty.
NB: most TC plastic plates will be dissolved by the acetone, but most 50ml Falcons should be ok, but check first.
2) Have a second empty 50ml falcon ready.
3) Drop about 10-20 individual coverslips one by one into the 50ml falcon with the Histogrip solution. Re-cap and invert tube gently several times.
4) Decant the Histogrip solution into the empty 50ml falcon.
5) Place coated coverslips into a 3rd Falcon full of TC clean H2O
6) Repeat steps 3-5 until you have coated enough coverslips
7) Remove H2O and wash coated coverslips 3x with H2O
8) Separate individual coverslips out onto large piece of clean Kimwipe or similar blotting paper and allow to air dry.
9) They can now be stored (for unto a year) in a 10cm plate or 50ml falcon.

General Buffers

PHEM Buffer
25 mM HEPES
1 mM EGTA
60 mM PIPES
2 mM MgCl2
pH = 6.9
(Add in this order.)

Antibody Blocking Solution (ABS)
1X PBS
3% BSA (or 5% Fetal Calf Serum)
0.1% Tween-20

Mix well and filter, aliquot and store at -20°C

Formaldehyde Fixation in PHEM buffer
(Good general use fixation, good for kinetochore proteins, ok for microtubules)

1.    Wash coverslips 2x with 1X PBS.
2 .   If staining a cytoplasmic protein or if you have high background then try a short pre-permeabilize of cells using 0.1-0.5% Triton in PHEM buffer 30 sec -1 min at room temperature (RT).
3.    Carefully fix cells with 3.7% Formaldehyde (fresh is best) diluted in PHEM + 0.5%      TritonX-100 for 10 min at RT.
4.    Wash 4x with 1X PBS at RT. Can be stored at 4°C for 2-3 days at this stage.
5.  Block coverslips for 15-30 min in ABS, then proceed to Antibody Staining

Fixation with -20°C Methanol
(Good for microtubules and most proteins)

1.  Wash coverslips 2x with 1xPBS
2.  Remove all PBS (but do not allow the cells to dry), and immediately add enough -20°C methanol to cover the coverslips. About 2-3ml if using a 6 well plate.
3.  Put the plate in a -20°C freezer for 5 min (NB: can be stored for weeks as long as you keep the coverslips covered in MeOH).
4.  Remove coverslips from MeOH, and rehydrate them in 1xPBS with 0.1-0.5% Triton-X-100 for 10-20 min
5.  Block coverslips for 15-30 min in ABS, then proceed to Antibody Staining.

Antibody Staining

1.    Incubate with ABS  for 15-30 min at RT.
2.    Incubate with 1°Ab diluted in Cell Blocking solution in moist chamber.
3.    Wash 3x 1X PBS-Tween for 5 min at RT.
4.    Incubate in 2°Ab +DAPI in PBS-T in moist chamber.
5.    Wash 3x 1X PBS-T 5 min at RT.
6  .  Mount with Prolong Gold or Mowiol mounting medium on clean glass slide.

Mowiol 4-88 Mounting Medium

Mowiol 4-88 is a high-quality mounting medium with good anti-fade characteristics. It hardens and matches the refractive index of immersion oil, and thus is particularly suited for this form of microscopy. Additional anti-fade (DABCO) is added to further retard photobleaching.

Mowiol 4-88 (Calbiochem; 475904), DABCO (Sigma; D-2522)

1.   Add 2.4g Mowiol to 6g glycerol and stir briefly with a pipette.
2.   Add 12ml dH2O and stir at room temp for several hours or overnight.
3.   Add 12ml 0.2M Tris (pH 8.5) and heat to 50oC for 1-2 hrs while stirring.
4.   When the Mowiol has dissolved, clarify by centrifugation @ 500 x g for 15mins.
5.   Add DABCO to 2.5% (0.72g), aliquot and store at -20oC. Bubbles can be removed by centrifugation. Aliquots can be stored for up to 2 weeks at 4°C or frozen to -20°C for months

How to Synchronise Mammalian Cells in Culture

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General Guidelines

These guides are written primarily for HeLa cells, but it should be possible to extend these to other cell lines with a bit of optimisation.

1) Make sure your cells are happy !
2) Aim for around 75-85% confluence at the time of release.
3) Try and keep everything close to 37ºC, including media, washing media, PBS, TC hood, centrifuges and avoid having the cells out of the incubator for long periods… i.e. work quickly and be organised before you start.
4) Avoid excessive amounts of media on the plate. Cells need to condition the media and the more volume the longer it will take. For a 10cm plate 5-7ml is ideal depending on the timing. Thus short releases (less than 12 hours) use 5ml, longer use 6-7ml.
5) Always try and make sure your cells are very well spread out on the plate.  Clumps and areas of very high or low density will reduce your synchrony.

G1/S Synchronisation
Here you will block cells in late G1 early S phase usually for around 18-28 hours. This time is roughly equivalent to how long the doubling time is for the cell line. For HeLa’s 20-24 hours is normally used. Cells will roughly take around 6 hours to complete S phase, 1-2 hours for G2, and around 1 hour for mitosis. Thus roughly:
S phase = 2-6 hours after release.
G2 = 7-8 hours
Mitosis = 9-10 hours
G1= 11+ hours

Single Thymidine
1) Seed up asynchronously growing cells on your desired plate. Your seeding will depend on when you plan to block the cells. You can block at the time of seeding, once they have resettled or the next day. Generally you will want about 50-60% density.
2) Add 2.5mM (Update we have dropped to using 1mM, best to do a check with your batch of HeLa’s to determine the optimal dose) Thymidine, which depletes the cells of deoxycytidine triphosphate. NB: Thymidine is not very soluble in water so make it up  to a stock [100mM] in PBS, and make sure you use PBS not water will need to add 25µl/ml of media.

3) Wait 20-24h, to arrest the majority of cells in G1/S. NB: you will always still have the odd few cells in mitosis… but thats ok.

4) Wash the cells 3x with pre-warmed PBS or Media. I have found that media sometimes gives slightly better results, although this is the more expensive option.

5) Add back fresh media, with 25µM 2′-Deoxycytidine (Santa Cruz #sc-231247), to replenish the depleted pools, and promote timely entry into S phase. NB some protocols suggest adding 25µM of Thymidine as well, this may help improve the releases slightly.

Double Thymidine
1) After the first release (step 4 above), wait 8-10 hours and then block cells again with 2.5mM Thymidine for an addition 16 hours.
2) Release as before (step 5 above).

NB: a double block is preferred when you require a very tight synchrony and time points from each different cell cycle phase for biochemistry. However, it does add a extra layer of complexity and if not done perfectly can result in a worse synchrony than a single block.

Hydroxyurea (HU)
Similar to thymidine although you use 2mM to block the cells. Also this can only be done as a single block, and no 2′-Deoxycytidine is needed in the release media.
The downside to the method is that some cell lines (e.g. U2OS) will start to over-duplicate their centrosomes if left in Hydroxyurea for too long.

Late G2 Block
This method is excellent for enriching cells in mitosis for either IF or movies.

RO-3306 (Cdk1 inhibitor)
Although a single block can work, best results are achieved by first blocking cells in G1/S with Thymidine or HU.
1) Release cells from G1/S as per instructions above.
2) Add 10µM of RO3306 to cells, ideally 4-6 hours post release to allow cells to get thru S phase.
3) Wait until the majority of cells have reached late G2. This is usually around 12h post release from G1/S.
4) Wash out the drug very well, with at least 3x washes with media or PBS.
5) Add back fresh media. Cells should begin entering Prophase within about 15-30 min of release. Metaphase peaks around 30-60 min and most cells should have completed anaphase/telophase by 90-120 min.

Here is a movie showing a best case synchrony, where around 90% of cells undergo mitosis. Typically, you should expect to see 50-60%.

Pro-Metaphase
This method is excellent for doing biochemistry on mitosis as it allows for highly enriched samples with a tight synchrony. It can also be used for movies if great care is take during the washing stage not to wash the cells off the plate. It is not suitable for IF.

Nocodazole
Like RO3306, you can use just a single  block with Nocodazole for 20-24 hours, but this will result in an increase in the level of death and better results are achieved by doing a pre-sync with thymidine or another G1/S blocker. NB: cells with a functional Chfr/Antephase checkpoint will delay for significant time during late G2 early prophase. HeLa cells do not have a Antephase checkpoint, but you may still notice a 1-2 hour delay in mitotic entry in response to Nocodazole.

1) After release from G1/S add Nocodazole at the desired concentration (25-3000ng/ml) ideally 4-6 hours after release, although you can add it straight away if you’re lazy. If you plan on releasing cells from the Nocodazole arrest then use 25-50ng/ml. If you are only interested in blocking cells use 100ng/ml. If you want to completely depolymerise microtubules then use 1-3µg/ml.
2)  Wait 12-14 hours after release, this should be sufficient for the majority of cells (80-90%) to block in Pro-Metaphase (P-M). You can easily see this by comparing the number of rounded up cells (P-M), to flat attached cells, likely G2.
3) For Biochemistry, recover media and floating cells from plate and put into a falcon tube. Bang the plate several times to help detach mitotic cells. Add a small amount of fresh (warm) media to recover detached cells. Repeat this once more. Check plate under a microscope that you still have interphase cells attached, and have recovered the majority of P-M cells.
4) You now have a nice highly enriched sample of pro-metaphase cells.
5) If you want to release, then gently spin cells down for 2-3 minutes at low speed (usually 1000rpm is sufficient). Remove media, gently resuspend pelleted cells with excess of fresh media (without any Noc in it). Repeat 1-2 times. NB: cells are very fragile at this stage, excessive washing can damage them leading to a poor release. Thus its a balancing act between washing away enough Noc to allow cells to recommence Mitosis. Generally cells will start arresting at doses of around 5ng/ml of Noc.
6) For movies, do not shake off the cells. Be very very very gentle with the plate, remove the media, and slowly add back fresh media. Repeat 2-3 times. Then immediately start filming your cells.

Complete Guide to Cell Cycle based Flow Cytometry

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A complete ‘ish’ guide to Flow cytometry or FACS for cell cycle work.

Covers all aspects from tissue culture, through to running and analyzing your samples on a FACScan or FACS Calibur.

It was written by me a few years ago and so is a bit out of date.  For example there is a new “prettier” version of CellQuest (Pro) now in use, but essentially it is still the same.

We will be at the Sydney Light Optical Users Meeting on July 24th 2014

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Great news, Cell Division Lab will be at the  Sydney Light Optical Users Meeting, hosted by Dr Pamela Young at Sydney University, this Thursday (24th of July).

I will be presenting a short seminar on “Imaging and Analysing Cell Division”.

If you would like to attend please contact Pamela asap. Her details are below!

Hope to see you there !

Sydney Light Optical Users Meeting July 2014

Anti-Oxaidants and Cancer…A complicated story!

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There has been a bit of press lately suggesting that Antioxidants might actually be bad for cancer… not good as they are commonly promoted in the media.
IFLS has put together a great article on some of the reasons why antioxidants might not be such a great thing [Link].

In addition, we recently wrote a review article about how different ‘stresses’ including oxidation can affect mitosis, and cancer. We also came to a similar conclusion in our review,  that antioxidants were a complicated and not always benifical for treating cancer. One of the main reason we suggested this was due to the fact that many common antioxidants are part of the Flavonoid family. On the surface that sound great, but many Flavonoids also happen to potently inhibit cyclin dependent kinases (Cdks). Coincidentally, our other recent article in Cell Cycle, showed that partial inhibition of Cdk1 can dramatically disrupt mitosis and drive severe cytokinesis defects and polyploidy (see video below). These mitotic defects are the foundation of chromosome instability  (CIN), which is a hallmark of more aggressive cancer types, that are also resistant to most chemotherapies and treatments. In simple terms, there is a possibility that in some cases, taking large quantities of dietary Flavonoids (e.g red wine, dark chocolate etc) could drive the formation of more aggressive cancers. This is definitely an area that needs a lot more research, and as always make sure that you fully discuss any dietary and supplements with your oncologist.

 

This is what happens when a ‘fairly normal’ cancer cell is treated with low doses of a Cdk1 inhibitor.

Here is a picture of a polyploid cancer cell, which was produced by partially inhibiting Cdk1.

ImageJ=1.48f unit=micron

 

Our Latest Review Article “Stressing Mitosis to Death” is now online !

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Great news, our latest review article “Stressing Mitosis to Death” has been accepted for publication by Frontiers in Oncology. You can access the provisional pre-press version here.

The review is about how common stresses affect mitosis, and the impact these stresses can have on the blockbuster mitotic chemotherapy drug Taxol (paclitaxel)

Finally here is one of the beautiful figures drawn by our own Sam Rogers for the Review!  Hope you enjoy the read !

 

Fig. v4