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 Null 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 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 Chart Basic Cell Culture

Basic Cell Culturing Procedure Draw out used medium from flask of MCF-7 cells. Add 5ml of hanks solution to flask and invert so hanks covers all of the bottom of the flask. Remove the hanks solution and then add 2ml of Trypsin and invert so solution covers all the of the bottom of flask. Put the flask of cells into the incubator for 4 minutes. 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. 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. Vortex test tube to ensure the cells are not attached to each other.

Procedure-(MCF-7) 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).

Procedure-(MCF-7)[continued] 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 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 in note book. 14. Repeat this process for 3 trials.

Procedure-(BT-20) 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] Add 20 micro liters of CellTiter 96 Aqueous One Solution Proliferation Assay (MTS) to each well. 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-(Ethyl Alcohol) Draw out used medium from flask of MCF-7 cells. Add 5ml of hanks solution to flask and invert so hanks covers all of the bottom of the flask. Remove the hanks solution and then add 2ml of Trypsin and invert so solution covers all the of the bottom of flask. Put the flask of cells into the incubator for 4 minutes. 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. 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. 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. 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) 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.

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