In order to make measurements of the cell cycle by 2ca6u9z

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									 Tiffany Friedman
 Scientists                                                             Scientists as Teachers, Teachers as
 Temple University
 0841377                                                                             NSF GK12- Award #

                Cell Cycle Analysis by Flow Cytometry
        Flow cytometry is the measurement of physical or
                                                                                                              1
chemical characteristics of a cell. In a flow cytometer cells flow
one at a time through an observation point where a laser
observes each cell and emits light responses. The light is
scattered by the cells into two forms, refracted light (due to lens
like properties of a cell) and reflected (off the internal surfaces),
giving us information regarding size and granularity of the cell.
In this way we are able to differentiate between the different
types of cells.



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                                                     Flow Cytometry can be used to see what part of the cell
                                             cycle a majority of a group of cells are in. In order to make
                                             measurements of the cell cycle, particular dyes that bind to
                                             DNA are used. Dye molecules bind to DNA binding sites and
                                             provide a direct relationship between the amount of dye and
                                             the amount of DNA. After flowing through a cytometer,
                                             corresponding software can produce histograms and dot plots
                                             to be used for analysis.



 3                         Histogram                                                Dot Plot                  4




       Tumor suppressors are proteins that suppress the ability of cancer to develop. One important
function of tumor suppressors is to control of the progression of a cell through the cell cycle. If cells lose
that control, they are able to synthesize damaged DNA before it is repaired or divide when the DNA is
damaged. The resulting daughter cells can pass on this damaged DNA to their progeny. This can result in
cancer.

        Flow cytometry is helpful when trying to see if particular tumor suppressor proteins are doing
their job. They are often inhibited from working by cancer causing agents!
Apply your knowledge:

        It is hypothesized that HBx, the hepatitis B protein that causes most of the problems within infected liver
         cells, downregulates the tumor suppressor p53.
        p53 causes cells to undergo apoptosis (cell death) when the cell’s DNA is too badly damaged to be
         repaired.
        You decide to test healthy liver cells (H), liver cells with OVER-expressed p53 (p53++), and liver cells
         infected with hepatitis B (Hbx) by flow cytometry.

Below are your dot plot results:




 5                                         6                                          7
                (H)                                    (p53++)                                     (HBx)
 1. According to the reference on the first page, in what phase are: (a) most of the
      healthy cells in? (b) The p53++ cells? (c) The HBx cells? Explain why these cells
      would be in these phases.




2. Does this data support the hypothesis? Why?




References:
1. http://users.path.ox.ac.uk/~nrust/diag1.gif
2. http://themedicalbiochemistrypage.org/images/cellcycle.jpg
3. J. Paul Robinson, Still from: Introduction to Cytochemistry II, Downloadable Lecture at: http://www.cyto.purdue.edu/class/
4. http://media.wiley.com/CurrentProtocols/CY/cy0923/cy0923-fig-0001-1-thumb.gif
5. Modified from reference #4.
6. Modified from: http://tools.invitrogen.com/content/sfs/gallery/high/A35137%20Web%20image.JPG
7. Modified from: http://www.nature.com/nrclinonc/journal/v6/n10/images/nrclinonc.2009.127-f1.jpg

								
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