d. Tutorial 4: Methods for Studying Cell Cycle Flashcards
Animal models are used to study the cell cycle. What is the advantage of studying drosophila?
after its fertilized all the developing cells divide synchronously (all cells progress through the cell cycle at the same rate)
What does it mean when cells divide synchronously?
it means that all the cells are at the same stage of cell cycle.
How does one examine the events that regulates the cell cycle transitions?
by sampling the cells at different times
Unfortunately, when analyzing the regulation of cell cycle transition in mammals the cells tend to divide ________.
a) at the same time
b) synchronously
c) asynchronously
d) too fast
e) too slow
c
What are the 2 general methods for synchronizing mammalian cells?
- adding a chemical to block a step, waiting for all the cells to reach that step, then removing the block
- withholding nutrients that is required to progress though the cell cycle
Describe the result of the following treatment on cells going through the cell cycle.
1. thymidine
2. hydroxyurea
3. colcemid
4. nocodazole
- arrest in the early S phase = blocks DNA synthesis by inhibiting the synthesis of dNTPs (mammals
- same as 1
- arrest in prophase of mitosis = disrupting microtubule formation preventing formation of mitotic spindles
- same as 3
how does one determine the cycle stage of a cell?
by measuring the amount of DNA present
How much DNA would be present in the following phases?
a) G1
b) S
c) G2
a) 1 copy (1C = diploid) = standard
b) 2 copies (2C = tetraploid) = due to replication
c) 2 copies = due to it being after replication but before division
What is the purpose of the flow cytometry tech?
measure the difference in copy number in order to distinguish the phase that that target cell is in
Describe the 6 steps to the flow cytometry tech being used to determine the cycle phase of a cell, including the following terms; fluorescent dye, propidium iodide, flow cytometer, capillary tube, laser beam, wavelength, light
- fix and label the cells using the fluorescent dye propidium iodide
- add the sample to the flow cytometer
- cells travel through a capillary tube past a laser beam
- when the laser hits cells labeled w/ a fluorescent dye of the correct wavelength is hit the cell emits light
- emitted fluorescence = amount of DNA in each cell
- use the amount of DNA to determine the cycle phase of that cell
What is the significance of propidium iodide?
its the type of fluorescent dye used to study the DNA copy number for cell cycle analysis
When fluorescently labeled cells enter the capillary tube of flow cytometry and encounter the laser beam describe what is being measured in the following cases
a) forward scatter
b) side scatter
c) emitted light
a) cell size
b) cell shape + inner complexity
c) amount of DNA
What is the significance of a cell cycle profile?
to plot the measurement of each cell after its gone through a flow cytometer
Flow cytometry can be used to analyze all of the following except
a) cell size
b) apotosis
c) cell shape
d) amount of DNA in a cell
e) all of the above
e
describe apoptotic bodies
where fragmented chromatin that is programmed to undergo apoptosis is encapsulated (the place where DNA that is going to die goes to die)
Describe each of the peaks shown in this flow cytometry graph. How much DNA is in each?
- apoptotic bodies = low amount of DNA
- G1 phase = 1 copy of DNA
- G2/mitosis phase = 2 copies of DNA
This image is showing a cell undergoing which phase
a) interphase
b) prophase
c) metaphase
d) anaphase
e) telophase
f) cytokinesis
a
This image is showing a cell undergoing which phase
a) interphase
b) prophase
c) metaphase
d) anaphase
e) telophase
f) cytokinesis
b
This image is showing a cell undergoing which phase
a) interphase
b) prophase
c) metaphase
d) anaphase
e) telophase
f) cytokinesis
c
This image is showing a cell undergoing which phase
a) interphase
b) prophase
c) metaphase
d) anaphase
e) telophase
f) cytokinesis
d
This image is showing a cell undergoing which phase
a) interphase
b) prophase
c) metaphase
d) anaphase
e) telophase
f) cytokinesis
e
This image is showing a cell undergoing which phase
a) interphase
b) prophase
c) metaphase
d) anaphase
e) telophase
f) cytokinesis
f
Examine the following plot produced by flow cytometry with cells stained with the DNA dye propidium iodide and answer the following questions.
a) True or False. This sample has more cells in G2 than in G1.
b) True of False. There are no apoptotic cells in this population.
a) false - We can see the higher peak is closer to 0 (further left on the x-axis), meaning that there are more cells in this group with only 1C of DNA or in G1.
b) true - We can see that there are only two clear distinct peaks representing cells with 1C and 2C. The lack of a broad flatter band with low propidium iodide signal suggests that there are no apoptotic bodies in this sample.
Imagine you treat cells with the drug colcemid and have effectively arrested 95% of your cells in prophase. What will the results from flow cytometry look like when cells are stained with propidium iodide? Draw out a figure like the one shown
Your results will look like this. Cells arrested in prophase will have two copies of DNA as they are past S phase. So you will see a large peak farther from zero representing the higher propidium iodide signal seen in cells with 2C. Because you will still see ~5% of cells in G1 you would also see a very small peak closer to zero representing a lower propidium iodide signal seen in cells with 1C.
