Protocols for mitosis movies

Part A – Treatment of cancer cell cultures with chemotherapeutic drugs

You will be provided with a culture plate containing six wells. Each well contains the HeLa cells we will be using in our study. The cells were added to the wells in the form of a suspension, and, as they sunk to the bottom of the well, they stuck to the plastic and started to divide. They were then incubated until the cells covered a large proportion of the plastic (confluence). In addition to being a place where the cells grow, each well will act as a treatment chamber and “slide” as the microscope we use can view the cells through the bottom of the well. The cells adhere quite strongly to the bottom of the wells. This means that the cell culture liquid can be replaced and the cells can be washed without any loss.

The plate provided contains cell culture media. As eukaryotic cells are more complex than bacterial cells, the medium used to grow them is more complex as well. This medium may consist of:

• Basal Medium – a commercial broth containing salts, sugars, buffers and other nutrients needed by all cell culture lines. These media are often coloured pink due to the presence of a pH indicator. As the cells metabolise, they produce wastes which change the colour of the medium to yellow, which can be used as an indicator of when the medium should be changed.

• Foetal Bovine Serum (FBS, also called Foetal Calf Serum) – the liquid component of clotted blood from a bovine foetus. This provides a complex series of nutrients required by the cultured cells.

• Pen/Strep/Glut – a mixture of the antibiotics penicillin and streptomycin, and the amino acid glutamine. The antibiotics prevent the growth of bacteria which may contaminate the medium and interfere with the growth of the cells. The glutamine supports the growth of rapidly growing cells.

• Sodium Pyruvate – pyruvate is the product of glycolysis and is the feedstock for the citric acid cycle in cellular respiration. It is included as an additional energy source.

• HEPES Buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) – a buffer which is particularly efficient in resisting the changes in pH caused by the release of carbon dioxide by cells

Unfortunately, the cell culture media used to grow these cells is incompatible with the optimal conditions for timelapse imaging of cells. Therefore, the cells must be washed and the medium replaced with another before proceeding.

Preparation of cell culture media, phosphate buffered saline and drug solutions

To prevent contamination, all of the following procedures must be carried out in the tissue culture hood.

To a 50ml centrifuge tube, add the following components :

Hams F12 Basal Medium	Serum Supreme
45mL	5mL

You are provided with phosphate buffered saline (PBS) at a concentration which is ten times (10X) more concentrated than what is needed – this is known as a stock solution. Stock solutions are made at a higher concentration and then diluted as necessary to provide working solutions (1X). The use of stock solutions saves us the bother of having to make up new solutions every time they are needed.

You need to make your working solution of PBS by diluting it appropriately in water. You are provided with a 100mL sterile “Baxter” water bottle for this purpose. The process for working out what volume to add to the Baxter water is as follows :

• Total volume     = 100mL.

• 1/10 of 100mL     = 100 ÷ 10  =  ____ mL.

• volume of 10X stock needed is ____ mL.

• Remove this volume from the Baxter water bottle.

• Add this volume of 10X PBS stock to the Baxter water bottle.

The Taxol is also provided as a 10X stock and will need to be diluted 1 in 10, however the amount needed is significantly less than the PBS.

• For our purposes, 10μL should be sufficient

• Total volume     = 10μL.

• 1/10 of 10μL     = 10 ÷ 10  =  _____ μL.

• volume of 10X stock needed is _____ μL.

• volume of culture medium needed is 10 - _____ μL = ______ μL

• Combine the calculated volumes of Taxol Stock and culture media in a labelled Eppendorf tube.

Treatment of cells

In the tissue culture hood :

• Remove the cell culture medium from each of the wells using the vacuum line

• Add 3mL of 1X PBS to each of the wells

• Remove the PBS from each well using the vacuum line

• Add 3mL of the prepared F12/Serum Supreme culture medium to each of the wells

• To each of the wells, add 3μL of the drug to be investigated. Leave one well untreated – this will be your negative control which should show how the cells divide in the absence of the drug. You will compare the results of your drug to this control when analyzing your results.

• Place the culture plate in the 37°C CO₂ incubator

Record which treatment was performed in each well in your manual.

Part B – Microscopy and analysis of results

Setting up and running the microscope

Your plate will be taken to the live cell imager located in the Ground Floor laboratories of UQDI. This consists of a microscope with a miniature incubator built into the stage. This incubator is the same as the larger ones which we use to grow the cells, maintaining them at a constant temperature of 37°C in an atmosphere containing 5% CO2.

This is an inverted microscope, meaning that instead of the lenses being placed above the specimen as in a normal microscope, the lenses are located beneath, closer to the bottom surface of the wells in the plate where the cells are found. Light is shone down through the culture plate and the lenses capture images of the cells stuck to the bottom.

When we place the plate on the microscope, we select five suitable fields in each well, programming these into the automated stage. The microscope will return precisely to each of these fields every time it takes a photograph, every 20 minutes.

Each time the microscope images a field, it takes two photographs. One image shows the cells under phase contrast a form of brightfield microscope that accentuates the contrast (animal cells lack cell walls and are difficult to see without staining). The second photograph is taken under blue light and shows the fluorescence signal generated by the GFP tagged histones. Note that while the light given off by the nuclei in this image is green, the strength of the signal is quite weak. A black and white camera is used to image both the brightfield and fluorescent fields as these are more sensitive to dim light than colour camera. The microscope’s software gives a false green colour to the fluorescent image and then combines the two images together (see Figure 4).

The microscope will take two images of each field, five fields to a well for all six wells, every 20 minutes over a period of 24 hours. When it is finished, the images will be processed and combined then downloaded and brought to you to make into a movie.

 

Making the movie

You will be provided with all of the combined images from all five fields in each well. It is then a relatively simple task to join all of these images together to create a movie. In the example below, we will be using Windows Movie Maker to do this.
 

 

Analysis of results

Your aim in this analysis is to observe any changes between the patterns of cell division between the untreated control cells and each of drug treated cells. Examples of difference which may be encountered include :

• Delays in entry into or departure from mitosis

• Unusual patterns of apoptosis

• Abnormalities during mitosis (eg. tri- or quadripolar spindles, failure of cytokinesis)

The easiest way to do this is to take a tally of cells exhibiting the trait you are looking for in each image and graph this as a value over time. You could do this analysis for every image individually, however in the interest of saving time (and eyestrain), you can use the movie you have made and select time points through it.

What to look for

Figure 5 shows series of images which follow a cell as it passes through mitosis :

From these images, it is relatively easy to see when a cell enters mitosis (it rounds up, forming the bright halo around it), mitosis is completed by the appearance of two equivalent anaphase chromosome bars, and cytokinesis (the bright membrane pinches off to form two daughter cells).

Apoptosis is a process of controlled cell death. When cells are damaged and they break down in an uncontrolled fashion, the cell contents are released into the surrounding tissue and these may affected other cells (sometimes triggering damage which causes more necrosis). In apoptosis, cell components are packaged up into discrete membrane-bound parcels which are then disposed of by immune cells without causing damage. Apoptosis is the method the body uses to dispose of cells which are damaged or otherwise faulty, or which are no longer needed (eg. removing the webbing between the fingers and toes during normal foetal development).

Apoptotic cells show nuclear staining as bright as mitotic cells but present as small discrete bundles (representing the nucleus being parceled up into membrane-bound bodies) and a distinct irregular, “bubbly” appearance to the cytoplasm and outer membrane (see Figure 6).

You will be recording the following information about each cell on the field :

• Frame at which the cell enters mitosis (ie. when it rounds up). This is a cumulative score – once a cell is counted it is part of the count permanently.

• Frame at which the cell leaves mitosis (ie. when mitosis is complete by the appearance of two anaphase chromosome bars). This is also a cumulative score.

• Time in mitosis (the first value above subtracted from the second)

• Does cytokinesis occur and is it normal i.e. giving two equivalent daughter cells

• Does apoptosis occur ? If so, when (frame number) and at what stage (before mitosis, during mitosis, after cytokinesis, etc)

Performing the analysis

• Progress through the movie frame by frame (you can do this using the movie, or you can use the raw images for this)

• You will follow each cell as it enters mitosis

• Record this information in a table or spreadsheet similar to the one in your manual.

Analysing the results

The simplest analysis to perform is to graph the cumulative percentage of cells in mitosis against time (frame). In untreated control cells, this should yield a roughly straight line (see Figure 7).

Any changes to mitosis will then be seen as a deviation from this relationship. Figure 8 shows a treatment which results in a delay in entry into mitosis.

A similar time series can be plotted for the cumulative number of cells which undergo apoptosis.

Delays in the time spent in mitosis can also be presented to compare the effects of different drugs. In the scatter diagram below (Figure 9), the time spent in mitosis has been plotted for control cells, as compared to two other drugs.

Conclusions

From a detailed analysis of the results, you should be able to answer the following questions :

• What differences are there between the progression of cells treated with each of the drugs tested and that of the untreated control cells ?

• Can you match these effects with the mode of action of each of these drugs described earlier ?

• Are there any drugs whose effects are similar to the unknown experimental drug ? If so, what category of drugs do you think this new drug belongs to ?

• Did you notice anything else occurring in the treated cells that was different to the control cells ? What do you think might have caused this ?

Further investigations

• What is the average time for these cells to complete mitosis ?

• We only provided you with minimal information about the modes of action of each of the drugs used. Research more information on each of these drugs (topics could include : structure of the drug, other drugs with similar activities, how extensively the drugs are used, what cancers they are used to treat). You could also research other chemotherapeutic drugs and predict what effect they would have on the cell cycle.

• Using images from your raw data, construct a poster showing the different stages of the cell cycle and mitosis.

• Through careful cropping of the control cell raw images, construct a movie showing just one cell passing through mitosis that could be shown to younger science students at your school. Use the editing function on Movie Maker to insert captions, a soundtrack and even a commentary.