Transcript WilliamsPJAS2008

The Effect of Bisphenol A on the
Growth of Brest Cancer Cells
Bisphenol A (BPA)
BT-20
MCF-7
Human tumor cell line.
The cell line was originally taken from a 69 year old lady who had a
breast tumor in 1970.
Has expressed estrogen receptors.
Found in the mammary gland in the human breast
It is designated as a epithelial type of cell
Well known as a useful model to study hormone-responsive breast
cancer as the cell line contain receptors for estrogen.
Estrogen both starts synthesis of certain proteins and increases rate of
proliferation.
MCF-7 cells tend to grow in colonies.
Very responsive to b-estradiol.
Numerous studies of nude mice have shown that MCF-7 cells are
capable of forming tumors.
Hypothesis
If MCF-7 (estrogen responsive) breast cancer cells are
exposed to different concentrations of Bisphenol A, then as
the concentrations go up so will the growth rate of the cells.
Null Hypothesis
If MCF-7 breast cancer cells are exposed to different
concentrations of Bisphenol A, then as the concentrations
will have no consistent effect on growth rate of the cells.
Materials
MCF-7 cell line
Bisphenol A
Hemocytometer
BT-20 cell line
Laminar Flow Hood
Bleach
Cell flasks
Plate Reader
Water
Petri Dishes
Microscope
Paper Towels
Pipettes (different sizes)
Plates
Plastic Gloves (non latex)
Pipette Man (different sizes)
Table
Lab Coat
Multiple Pipette Man
Pen
Electronic Scale
Repeating Pipetter
Notebook
Chemical Spatula
Medium
Ethyl Alcohol
 Test Tubes
Hanks (HBSS)
MTS
Computer
Trypsin
Trypan Blue
Calculator
Timer
Alcohol wipes
37º C water bath
Plastic Containers
96 well plate
15ml conical tube
Procedure- Flow Chart
Basic Cell Culture
Basic Cell Culturing Procedure
1. Draw out used medium from flask of MCF-7 cells.
2. Add 5ml of hanks solution to flask and invert so hanks covers all of the
bottom of the flask.
3. Remove the hanks solution and then add 2ml of Trypsin and invert so
solution covers all the of the bottom of flask.
4. Put the flask of cells into the incubator for 4 minutes.
5. Remove flask from the incubator and look at cell under the microscope
to make sure all as the cells have detached from the bottom of the flask.
6. Add 5ml of fresh medium to the flask and pipette (about 7ml) of solution
out of flask and put it into a plastic test tube.
7. Vortex test tube to ensure the cells are not attached to each other.
Procedure-(MCF-7)
1. After vortexing the cell solution for 30 seconds, draw out 36 micro
liters and pipette into a well.
2. Then pipette 36 micro liters of Trypan blue cell dye into the well
with the cell solution and mix the new 72 micro liter solution
thoroughly.
3. Draw up enough solution to fill the hemocytometer and count the
number of cells.
4. After using calculations to figure out theoretical number of cells
in the cell solution, make a solution of 20ml of medium and cell
solution so that each well in a 96 well plate will have 5000 cells
(50,000 cells/plate).
5. Vortex 20ml solution for thirty seconds and then pipette the
solution into wells (in each column only rows
D to H).
6. Incubate plate for 24 hrs
7. Treat plates with the solutions of ethyl alcohol
and Bisphenol A (BPA) solution that was
made (see BPA concentration page).
Procedure-(MCF-7)[continued]
8. Incubate the plate for 72 hours.
9. Remove the used medium from the plate and add 100 micro
liters of fresh medium to each well of the plate.
10. Add 20 micro liters of CellTiter 96 Aqueous One Solution
Proliferation Assay (MTS) to each well.
11. Return plate to incubator for 2 hours.
12. Take plate from the incubator and check for any air bubble that
may of formed, and pop the bubbles if any have formed.
13. Put plate in plate reader and record the absorbance of each well
in note book.
14. Repeat this process for 3 trials.
Procedure-(BT-20)
1.
2.
3.
4.
5.
6.
7.
8.
9.
After vortexing the cell solution for 30 seconds, draw out 36
micro liters and pipette into a well.
Then pipette 36 micro liters of Trypan blue cell dye into the well
with the cell solution and mix the new 72 micro liter solution
thoroughly.
Draw up enough solution to fill the hemocytometer and count
the number of cells.
After using calculations to figure out theoretical number of
cells in the cell solution, make a solution of 20ml of medium
and cell solution so that each well in a 96 well plate will have
5000 cells (50,000 cells/plate).
Vortex 20ml solution for thirty seconds and then pipette the
solution into wells (in each column only rows D to H).
Incubate plate for 24 hrs
Treat plates with the solutions of ethyl alcohol and Bisphenol A
(BPA) solution that was made (see BPA concentration page).
Incubate the plate for 72 hours.
Remove the used medium from the plate and add 100 micro
liters of fresh medium to each well of the plate.
Procedure-(BT-20) )[continued]
10.
11.
12.
13.
14.
Add 20 micro liters of CellTiter 96 Aqueous One Solution
Proliferation Assay (MTS) to each well.
Return plate to incubator for 2 hours.
Take plate from the incubator and check for any air bubble that
may of formed, and pop the bubbles if any have formed.
Put plate in plate reader and record the absorbance of each
well.
Repeat this process for 3 trials.
Procedure-(Ethyl Alcohol)
1. Draw out used medium from flask of MCF-7 cells.
2. Add 5ml of hanks solution to flask and invert so hanks covers all of the
bottom of the flask.
3. Remove the hanks solution and then add 2ml of Trypsin and invert so
solution covers all the of the bottom of flask.
4. Put the flask of cells into the incubator for 4 minutes.
5. Remove flask from the incubator and look at cell under the microscope
to make sure all as the cells have detached from the bottom of the flask.
6. Add 5ml of fresh medium to the flask and pipette (about 7ml) of solution
out of flask and put it into a plastic test tube.
7. Vortex test tube to ensure the cells are not attached to each other. After
vortexing the cell solution for 30 seconds, draw out 36 micro liters and
pipette into a separate well.
8. Then pipette 36 micro liters of Trypan blue cell dye into the well with the
cell solution and mix the new 72 micro liter solution thoroughly.
Procedure-(Ethyl Alcohol)
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Draw up enough solution to fill the hemocytometer and count the
number of cells.
After using calculations to figure out theoretical number of cells in
the cell solution, make a solution of 20ml of medium and cell solution
so that each well in a 96 well plate will have 5000 cells (50,000
cells/plate).
Vortex 20ml solution for thirty seconds and then pipette the solution
into wells (in each column only rows D to H).
Incubate plate for 24 hrs
Treat plates with the solutions of ethyl alcohol and Bisphenol A (BPA)
solution that was made (see BPA concentration page).
Incubate the plate for 72 hours.
Remove the used medium from the plate and add 100 micro liters of
fresh medium to each well of the plate.
Add 20 micro liters of CellTiter 96 Aqueous One Solutio Proliferation
Assay (MTS) to each well.
Return plate to incubator for 2 hours.
Take plate from the incubator and check for any air bubble that may of
formed, and pop the bubbles if any have formed.
Put plate in plate reader and record the absorbance of each well.
Repeat this process for 3 trials.
Data
Data