The Interactions of Red Clover with Tamoxifen
Materials and Methods:
Materials:
- Cells (COS-7 African Green Monkey kidney cells)
- Red clover (in gelatin capsules)
- Ethanol [95% 9v/v)]
- Water
- 10% FBS- control
- 2% FBS- control (no Tx) (no E2)
- 2% FCS + 0.1, 1.0, and 10.0nM E2
- Cells (MCF-7 Human Breast Cancer Cells)
- 2% FBS + 0.1, 0.5, 1.0, 5.0, 10.0nM Tx
- 2% FBS + 10^-2 – 10^-6nM Red Clover
- 2% FBS + 0.1, 0.5, 1.0, 5.0, 10.0µM Tx
- 2% FBS + 0.1, 0.5, 1.0, 5.0, 10.0µM Tx w/ E2
- 2% FBS + 2.0µM Tx
- 2% FBS + 2.0µM Tx + 1:10^4 and 1:10^5 red clover
- 2% FBS + 4.0µM Tx
- 2% FBS + 4.0µM Tx + 1:10^4 and 1:10^5 red clover
- 2% FBS + 2.0µM Tx w/ 1nm E2
- 2% FBS + 2.0µM Tx + 1:10^4 and 1:10^5 red clover w/ 1nm E2
- 2% FBS + 4.0µM Tx w/ 1nm E2
- 2% FBS + 4.0µM Tx + 1:10^4 and 1:10^5 red clover w/ 1nm E2
Method:
Lab #1: Cell Culturing
COS-7 cells were visualized using an inverted phase contrast microscope. Cells were estimated to be ~90% confluent (density) on the culture plates. Medium was aspirated using the vacuum pump. Two milliliters (mL) of Trypsin Solution were added to cell culture plate to hydrolyze (digest) protein bonds that attach the cell to the plastic. Cells were incubated for three minutes. Cells were viewed under inverted phase contrast microscope to determine the progress of lifting. Using a 5 mL pipette, cells were relocated, “lifted,” from the plates to 15 mL sterile tubes containing 10 mL of the complete growth medium. Complete growth medium will help to inactivate the Trypsin solution, otherwise over-digestion of cellular proteins may result in reduced cell viability and re-attachment to cell plates. Cells were then centrifuged for 4 minutes at ~1000x G force (g) to pellet the cells, and the Trypsin/medium mixture was aspirated, and the cells were re-suspended in 10 mL of fresh growth medium using a 10 mL sterile pipettes. One mL of the cell suspension was added to a new cell culture plate containing 9 mL of fresh growth medium. This dilution (1:10) will allow the cells to have room to grow and divide on the plate. Cell plate was then placed in the incubator (at 37°C).
Lab #2: Cell Counting
Cells were lifted using the method outlined in Lab # 1 (see page 1). However, cells were re-suspended in 5ml, opposed to 10mL of medium. Cells were then viewed under the inverted phase contrast microscope, and counted using a hemocytometer. The total number of cells was approximated using the correct formula, as well as the number of cells re-plated. From the cells suspended in 5mL of medium, only 1ml was re-plated. Cells were re-plated to allow them room to grow, and to increase the cells overall viability.
Lab #3: Red Clover
A red clover capsule was opened, and the powder inside the gelatin capsule was allocated to four 1.5 mL tubes using the scoopula. Water (approximately 1 mL) was added to two tubes, and they were labeled accordingly. Ethanol was added (again approximately 1 mL) to the two remaining tubes, which were labeled as well. One tube containing red clover and water, and one containing red clover and ethanol were shaken by hand, and using the vortex in an attempt to make a solution. The two solutions were placed on two separate, clearly labeled slides, and observed using the inverted phase contrast microscope. Observations were recorded. The two remaining tubes (one with red clover and water, the other with red clover and ethanol) were mixed using the vortex, and then centrifuged for approximately five minutes. The liquid at the top of the two tubes was aspirated and placed in two separate and labeled slides and observed using the inverted phase contrast microscope. Observations were recorded.
Lab #4: Counting MCF-7 Cells
Culture plates were prepared of each condition. Victoria’s cells were seeded at 50 000 cells/well and Shalini’s cells were seeded at 25 000 cells/ well. On day 1 cells were treated with E2 and cells were counted. Cells were then counted in duplicate every 2 days for 10 days. In order to do so, cells were Trypsinized in 500µL of Trypsin. Cells lifted completely and were then added to a 1.5 mL tube. The cells were then spun down in a microcentrifuge and were then re-suspended in 100µL of PBS. Ten µL of the solution was added to the hemocytometer, cells were then counted, and counts were recorded.
Lab #5: Counting Treated MCF-7 Cells
Culture plates were prepared for each condition. Both sets of cells were seeded at 50 000 cells/well. Cells were then counted in duplicate every 2 days for 8 days. In order to do so, cells were Trypsinized in 500µL of Trypsin. Cells lifted completely and were then added to a 1.5 mL tube. The cells were then spun down in a microcentrifuge and were then re-suspended in a varying dilution of PBS. Ten µL of the solution was added to the hemocytometer, cells were then counted, and counts were recorded.
Lab #6: Tamoxifen and Estradiol
Culture plates were prepared for each condition. Both sets of cells were seeded at 50 000 cells/well. Cells were then counted in duplicate every 2 days for 8 days. In order to do so, cells were Trypsinized in 500µL of Trypsin. Cells lifted completely and were then added to a 1.5 mL tube. The cells were then spun down in a microcentrifuge and were then re-suspended in a varying dilution of PBS. Ten µL of the solution was added to the hemocytometer, cells were then counted, and counts were recorded.
Lab #7: Red Clover, Tamoxifen and Estradiol
Culture plates were prepared for each condition. Both sets of cells were seeded at 25 000 cells/well. On day 2, the cells were pre-treated with red clover. This treatment was left to settle until day 6. On day 6, we treated the cells with Tamoxifen, re-treated with red clover, and we applied Estradiol to the second set of cells. This was done so as to replicate the environment of the human female body. Our rationale for pre-treatment with red clover was to replicate the human body. As red clover is a common alternative type of medication, women would most likely be using it before and during their cancer treatments. Therefore, pre-treating was the best method to replicate this aforementioned cell environment. Also, it was determined in lab # 5 that red clover has a delayed effect upon MCF-7 cells. In other words, the cells only began to experience the estrogenic effect at approximately day 6 and day 8. Cells were then counted in duplicate every 2 days, starting at day 6 for 8 days following. In order count the cells they were Trypsinized in 500µL of Trypsin. Cells lifted completely and were then added to a 1.5 mL tube. The cells were then spun down in a microcentrifuge and were then re-suspended in a varying dilution of PBS. Ten µL of the solution was added to the hemocytometer, cells were then counted, and counts were recorded.