The Effect of Bisphenol A on the Growth of Brest Cancer Cells

Bisphenol A (BPA)

BT-20

MCF-7Human 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 cellWell 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 HypothesisIf 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.

MaterialsMCF-7 cell line

BT-20 cell line

Cell flasks

Petri Dishes

Pipettes (different sizes)

Pipette Man (different sizes)

Multiple Pipette Man

Repeating Pipetter

Medium

Hanks (HBSS)

Trypsin

96 well plate 15ml conical tube

Bisphenol A

Laminar Flow Hood

Plate Reader

Microscope

Plates

Table

Pen

Notebook

Ethyl Alcohol

MTS

Trypan Blue

Timer

37º C water bath

Hemocytometer

Bleach

Water

Paper Towels

Plastic Gloves (non latex)

Lab Coat

Electronic Scale

Chemical Spatula

Test Tubes

Computer

Calculator

Alcohol wipes

Plastic Containers

Procedure- Flow ChartBasic 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. 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).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. 14. Repeat this process for 3 trials.

Procedure-(BT-20) )[continued]

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.

9. Draw up enough solution to fill the hemocytometer and count the number of cells.10. 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).

11. Vortex 20ml solution for thirty seconds and then pipette the solution into wells (in each column only rows D to H).

12. Incubate plate for 24 hrs 13. Treat plates with the solutions of ethyl alcohol and Bisphenol A (BPA) solution that

was made (see BPA concentration page).14. Incubate the plate for 72 hours.15. Remove the used medium from the plate and add 100 micro liters of fresh medium

to each well of the plate.16. Add 20 micro liters of CellTiter 96 Aqueous One Solutio Proliferation Assay (MTS)

to each well.17. Return plate to incubator for 2 hours.18. Take plate from the incubator and check for any air bubble that may of formed,

and pop the bubbles if any have formed.19. Put plate in plate reader and record the absorbance of each well. 20. Repeat this process for 3 trials.

Procedure-(Ethyl Alcohol)

Data

Data