Materials and Methods Cell Culture Two cell pellets, one with breast cancer cells (Line A) and

one with normal breast cells (Line B) were plated under cell media containing fetal bovine serum and 0.99 M Tamoxifen (TAM) and under a control media containing fetal bovine serum and ethanol. The cells were incubated for seven days at 37 C in air containing 5% carbon dioxide. Crystal Violet Assay A crystal violet assay was performed on the cells. The media was aspirated from the cells and 1% glutaraldehyde (Fisher Scientific) was added to each well. The cells were incubated for 15 minutes at room temperature. Glutaraldehyde was removed and 0.1% crystal violet (Fisher Scientific) was added to the cells. After ten minutes, the crystal violet was removed and 0.2% Triton X-100 (Fisher Scientific) was added. The cells were shaken for 15 minutes and then transferred to a new well plate. The absorbance of the cells was recorded at 590 nm with a SpectraMax M5 plate reader (Analytical Technologies). Measurement of Caspase Activity Cells (line A and line B) were pelleted and plated in extracellular matrix (ECM) attached or detached conditions. The cells were lysed with chilled caspase assay lysis buffer. The cells were incubated for 10 minutes on ice and centrifuged in a microfuge at 12,000 RPM, for 3 minutes at 4 C. Reaction buffer containing DTT was added to the supernatant of each sample and DEVDpNA was added after. A blank of reaction buffer, lysis buffer, and DEVD- pNA(p-nitroanilide) was prepared. The samples and the blank were incubated at 37 C for 35 minutes. The samples were transferred to a plate containing the standards and the absorbance was recorded for the samples at 405 nm on a microplate reader. A pNA standard curve was prepared by measuring the absorbance of light of DEVD- pNA concentrations at 0 M, 25 M, 50 M, 100 M, and 200 M. Measurement of Apoptosis To isolate the DNA from cultured cells, lysis buffer was added to each sample and incubated at room temperature for 10 minutes. Isopropanol was added. The sample was washed and filtered three times. Elution buffer and RNAse were added to the samples and incubated for 20 minutes at room temperature. A DNA gel electrophoresis was run on the cells with a 100 bp ladder. DNA was visualized afterwards. Results Crystal Violet Assay

The crystal violet assay demonstrated the absorbance of light at 590 nm of each cell line in EtOH and TAM. In cell line A, there was a high absorbance in EtOH and a low absorbance in TAM. In cell line B, there was a high absorbance in EtOH and TAM. (Fig. 1)

Fig.1 Crystal Violet Assay 0.1% crystal violet was added to the cells. After ten minutes, the crystal violet was removed and 0.2% Triton X-100 was added. The cells were shaken for 15 minutes and then transferred to a new well plate. The absorbance of the cells was recorded at 590 nm. The crystal violet assay determined how much cell growth occurred in each cell line base on the absorbance of the crystal violet dye. Caspase Activity A pNA standard curve was created to correlate the absorbance at 405 nm in each concentration of pNA. A higher UV absorbance indicates higher amounts of pNA in the cells (See Fig. 2). A bar graph was created of the absorbance of each cell line (A and B) attached or detached with 98M of pNA added to all the cells. (See Fig. 3).

Fig. 2 PNA Standard Curve A pNA standard curve was prepared by measuring the absorbance of light of DEVD- pNA concentrations at 0 M, 25 M, 50 M, 100 M, and 200 M.

Sample Absorbance
1.2000 1.0000 0.8000 0.6000 0.4000 0.2000 0.0000 A att a det b att b det

Series1

Fig. 3 Absorbance of attached and detached cell line A and B The cells lines A and B were transferred to a plate in which 98M of pNA added to all the cells. The absorbance was recorded for the samples at 405 nm on a microplate reader and compared to the pNA standard curve. Measurement of Apoptosis Activity The presence of apoptosis in cell line A and B was identified by running a DNA gel electrophoresis. Perhaps we did not isolate our DNA correctly as the DNA from the cells lines

could not be seen on the gel. Ideally, the DNA fragments would be seen in intervals of 200 bp (See Fig. 4).

Fig. 4 DNA Gel Electrophoresis A DNA gel electrophoresis was run on the cells with a 100 bp ladder. The DNA gel was visualized afterwards. However, the DNA in our cell lines cannot be seen in this gel.