Download Cell Cycle Biol 459B Bioimaging 1 The Mammalian Cell Cycle

Cell Cycle
Biol 459B Bioimaging
The Mammalian Cell Cycle
Objective: the goal of this exercise is to study the mammalian cell cycle using
quantitative light microscopy.
Introduction:
The ability to grow and duplicate is a fundamental property of life. Mammalian
cells require about 1 day, 24 hours, to traverse the entire cell cycle. From visual
examination of cells, the cell cycle can be divided into mitosis (M) when cells
physically segregate the chromosomes and divide into two daughter cells and the
remainder, or interphase (I). Additional experiments showed that cells replicate
their DNA during a discrete time in interphase. This phase is called S phase, for
DNA synthesis. The period prior to S is called G1 (gap 1) and the period after S,
but before M, is called G2 (gap 2), see Figure 1.
Figure 1. Schematic diagram of the cell cycle. From Alberts et al., MBOC5
Some cells exit the cell cycle and cease to proliferate. This phase of the cell
cycle is sometimes referred to as G0. Examples of cells that have exited the cell
cycle include most neurons and RBCs. Other cells do not divide all the time, but
can be induced to divide, for example by injuries to tissues or during an immune
response.
In this lab we will begin to explore the cell cycle using mammalian cells. Once
you have learned how to measure various features of the cell cycle, you can
design experiments to determine the factors that influence the cell cycle.
Materials:
Mammalian cells stained with DAPI and anti-tubulin antibodies
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Cell Cycle
Biol 459B Bioimaging
1. Identify and quantify the phases of mitosis and determine the mitotic
index.
Using the slide stained for DAPI and tubulin, you will determine the mitotic index,
which is defined as the percentage of cells in a population that are in mitosis.
You will also identify the stage of mitosis for all the mitotic cells that you observe.
Take images of fields of view at 40X. Collect at least 15 fields of view. Image
each field in DAPI AND fluorescein (microtubule) fluorescence. To take
representative fields of view, position the coverslip/slide on the microscope so
that you begin observation at one side; move the slide in a pattern (across, down,
across etc), so that you don’t quantify the same region of the coverslip twice.
Save the images. Count the total number of cells in each image and the number
that are in mitosis. Put the data in the Table. For each mitotic cell, determine if
the cell is in prophase, prometaphase, metaphase, anaphase, or telophase. Use
the diagram below to help you identify the stage of mitosis (prophase, nuclear
envelope is intact, chromosomes condensed; prometaphase, nuclear envelope
has broken down, but chromosomes are not aligned at the metaphase plate;
metaphase, all chromosomes aligned between the two spindle poles; anaphase,
chromosomes moving to the spindle poles; and telophase, chromosomes
beginning to decondense, cell undergoing cytokinesis. For reference, a
diagrammatic view of the stages of mitosis is shown in Figure 2.
2. Quantify the mitotic index.
Figure 2. Schematic of the cell cycle, showing the stages of mitosis. From Alberts, et al.,
MBOC5.
From the data, determine the MI and determine the % of mitotic cells that are in
each of the phases of mitosis. How many cells do you need to count to get
statistically meaningful data? Combine the data from all the groups: bring your
data to the computer at the front of the lab and enter it in the spread sheet.
What factors might alter the MI of a population of cells? From the data on stage
of mitosis, can you estimate which of the mitotic phases takes the longest time to
complete?
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Cell Cycle
Biol 459B Bioimaging
2. Measure the quantity of DNA in interphase cells.
When S phase is completed, each nucleus contains twice the DNA content of a
cell that has not undergone DNA synthesis. For this experiment, you can collect
images of fields of view at low magnification (or use the images that you just
collected). Using Image J, determine the integrated fluorescence intensity in
each nucleus. Measure at least 20 nuclei. Are all the nuclei the same size?
Bring the data to the computer at the front of the room and enter it in the spread
sheet. When all the data has been entered, generate a histogram or graph. Can
you determine which nuclei have undergone S and which have not?
3. Quiescent cells. Mammalian cells will enter a quiescent phase, G0, if serum is
withdrawn from the culture media. Obtain a coverslip that was serum starved for
24 hours and then stained with DAPI. Collect images of at least 15 fields of
view; save them. Determine the mitotic index and determine the percentage of
cells that have undergone DNA synthesis. (if you are running short on time, you
can do the measurements on the saved images at a later time). Compare the
data from these quiescent cells with the cells that were grown in serum (from part
1). Make another table with your data, and enter it into the class spread sheet.
What do you think might happen if you stimulated the starved cells with serum?
4. Cells arrested in mitosis. Microtubules are required for assembly of the
mitotic spindle and chromosome segregation during mitosis. What do you think
might happen if the assembly of microtubules was prevented? Obtain a slide
that has been treated with nocodazole for 6 hours, and then fixed and stained for
DAPI. Collect images of at least 15 fields of view. Measure the mitotic index.
Determine the percentage of cells that have undergone DNA synthesis. Make
another table with your data, and enter it into the class spread sheet. Determine
the stage of mitosis for each mitotic cell. At what point in mitosis do the cells
arrest?
Table 1: Mitotic Index and Stage of Mitosis
Field of
View
Total #
cells
#
mitotic
cells
Stage of Mitosis
Pro.
Promet
1
2
3
etc
3
Meta.
Ana.
Telo.