NIH3T3 Cells Lab Report
The samples were boiled for 3 minutes and centrifuged for 20 seconds. A polyacrylamide gel was assembled and the samples were loaded into it. The gel ran for approximately 45 minutes at 160 V. A western blot analysis was done in order to examine the Erk1/2 phosphorylation of the NIH3T3 cells. Three pieces of Whatman paper were wet in transfer buffer and placed on the anode plate. A wet PVDF membrane was placed on top of the Whatman paper. The gel was removed from holder and the stacking gel was removed. Forceps were used to place the gel onto the wet membrane and another Whatman paper was placed on top of the. The cathode plate was placed and started the transfer. Then the membrane was placed in TBST containing 5% milk for 60 minute while shaking. The membrane was rinsed twice with TBST (no milk) and placed in the primary antibody. The membrane was washed three times for two minutes
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each with 10 mL of TBST. The membrane was then placed in the secondary antibody and incubated for 30 minutes while shaking. The washing with TBST was repeated. It was taken to a dark room to expose the film and develop the image. Students received a 10 cm plate with Ras-transformed NIH3T3 cells and performed the same experiment that was done to find the cell density of non-transformed NIH3T3 cells. Then the students received a 6-well plate containing parental and Ras-transformed cells that had been starved for 24 hours in 2mL of media with 0.5% calf serum. The parental cells occupied the top left and middle wells and the Ras-transformed cells occupied the bottom left and middle wells. The students added 400 µL of calf serum (20%) and incubated the wells for 20 minutes. The culture medium was removed and cold PBS was used to wash the wells. The plate was immediately put on ice and 250 µL of cell lysis buffer was added to each well. The cells were scraped and the supernatants were transferred to Eppendorf tubes and incubated on ice for 20 minutes and centrifuged at 12,000 rpm in a cold room for 20 minutes. The supernatants were transferred to new Eppendorf tubes and put in a -20° C freezer. An SDS-PAGE and western blot were done for these cells in the same way they were done for the non-transformed cells (Dong).
Results:
The last page of the lab report contains the data from the experiment. Figure 1.1 shows the cell density of NIH3T3 cells. A hemacytometer was used to count these cells. The four numbers were averaged and multiplied by 2 in order to compensate for the dilution factor. This number was then multiplied by 10,000 in order to convert the units to cells/mL. These cells grew for one week before counting. Figure 1.2 shows the relationship between the concentration of the serum and the number of cells. As the results indicate, the cell density increases as the concentration of FBS increases. The cell density more than tripled when the FBS concentration increased from 2% to 10%. The cells were able to proliferate more in higher FBS concentrations because they received more nutrients that allowed them to survive and grow. Figure 1.3 shows the results from the SRB assay that was used to determine cell proliferation of the NIH3T3 cells. The SRB measures the absorbance of the cells, which can be used to determine the protein concentration. These results yielded similar results to the trend of increasing FBS concentrations. Furthermore, since these cells were given more time to proliferate their density was higher. Figure 1.4 shows the number of Ras-Transformed cells in each of the different concentrations of the serum. These numbers are extremely similar, which shows that the Ras-transformed cells are not dependent on the nutrients provided from the serum. This shows that the Ras-transformed cell density is independent of the concentration of serums or growth factors. Figure 1.5 shows the results from the SRB assay that was used to determine cell proliferation of the Ras-Transformed cells. This assay was similar to the one performed in Figure 1.3, the only difference was the type of cells examined. These results further proved that the Ras-Transformed cells grow independently of growth factors and nutrients because the absorbance and protein concentrations exhibited minimal change when the concentrations of FBS were changed. Figure 1.6 shows the western blot analysis performed on NIH3T3 fibroblast cells. This western blot was performed to examine the Erk ½ pathway of NIH3T3 cells without serum or growth factors, with calf serum, and with FGF. The control is lane 4, the cells with calf serum is lane 5, and the cells with FGF are in lane 6. This shows that the concentration increases greatly upon the addition of calf serum since that line is much darker than the control. The concentration should have also increased upon addition of the FGF, however it is evident that it did not. This is most likely due to the fact that the tubes containing FGF were old and the growth factors were no longer active. Figure 1.7 shows the western blot analysis performed on Ras-Transformed cells. This western blot was performed to examine the Erk ½ pathway of the Ras-Transformed cells without serum or growth factors, with calf serum, and with FGF. As is evident from the image, the addition of these substances that had increased proliferation in the non-transformed cells had little to no effect on the Ras-Transformed cells. This further proves that transformed cells are not reliant on the calf serum or FGF.
Discussion:
The purpose of this lab was to analyze the growth factor dependence of normal NIH3T3 cells and Ras-Transformed cells.
The goal of this experiment was to use cell cultures to monito the growth of these cells when treated with different calf serum and FGF concentrations. SRB protein assay was conducted in order to observe the cell proliferation. Western blotting was used to look at the relationship between the serum and activation of the Erk ½ pathway along with protein concentration. Thus, there were two hypotheses being tested during this experiment. This first hypothesis states: If various concentrations of Fetal Bovine Serum were added to non-transformed and transformed cells, the non-transformed cell density would increase with the concentrations of these substances and the transformed cells would be unaffected. The second hypothesis being tested is that the protein concentration of the Ras-transformed cells should also remain unaffected by the addition of serums and growth factors. However, the non-transformed cells will again increase with the concentrations of these
substances. The results obtained from this experiment support these hypotheses. The cell density of NIH3T3 increased greatly as the concentration of the calf serum increased. The density of Ras-Transformed cells, however, were unaffected by the addition of calf serum. This is because transformed cells are not reliant on nutrients to divide. The western blot looked at the protein concentration at different concentrations of the calf serum and FGF. It was concluded that the serum did affect the Erk ½ activation of the NIH3T3 cells. This is evident because the protein concentrations of the treated cells was higher than that of the control group, with the exception of FGF because it was possibly inactive. The Ras-Transformed cells, however, were not affected by these factors. Thus, the protein concentration remained constant as seen from the western blot. Since the serum did not affect the protein concentration, it is evident that the calf serum and FGF had no effect on the Erk ½ activation in the transformed cells. These results were expected because the non-transformed NIH3T3 cells have a finite life span and are dependent on calf serum for growth and survival. These cells grow by means of density-dependence inhibition, serum dependence, and/or anchorage dependence. The Ras-Transformed cells do no grow by these means and are able to proliferate infinitely independently of calf serum and growth factors. These results support the hypothesis. This experiment is significant to today’s society because of the rising cases of cancer. Cancer has affect nearly every person in some war. This experiment helps us to understand the difference between normal cell and transformed (cancer) cells. The growth of cancer cells can be analyzed by looking at the Erk ½ phosphorylation. This will provide an explanation of why and how cancer cells can grow at an increased rate. This knowledge can potentially provide new treatments and possibly a cure for cancer. A few potential problems with this lab could have been either human or experimental error. A human error could have been not incubating the cells for a long enough period of time after addition of trypsin. This would have resulted in some cells being attached to the plate and the overall cell count being lower than expected. The cell count could also be affected when counting the cells with the hemacytometer since the human eye is not fool-proof and may not have seen all of the living cells under the microscope. Another human error could be that the plate was not immediately put on ice after washing with PBS. This would also negatively alter the results. Furthermore, had some of the sample been lost when loading the wells of the gel, the western blot results could be altered. Aside from human error, experimental error could have also altered the results. For example, the FGF tubes that were used for the western blot analysis of non-transformed cells were old. This could be the reason why the sample with FGF was not visible on the western blot. If the FGF was inactive because of the age of the tube, it would not have induced growth of the cells like it should have. Furthermore, while transferring the samples and cells with the micropipette some of the cells may have remained in the tip. This would have lowered the cell count since solution would have been lost. Also, if the lab hood ventilation was not working properly that cultures would have been contaminated from outside air. In order to make this experiment more effective, an alternative approach could be used. Different types of cells could possibly be used. If the experiment involved epithelial cells, HeLa cells for example, the experimenter could compare the results from the different types of cells. Another alternative method could be the use of fluorescence instead of the western blot
each with 10 mL of TBST. The membrane was then placed in the secondary antibody and incubated for 30 minutes while shaking. The washing with TBST was repeated. It was taken to a dark room to expose the film and develop the image. Students received a 10 cm plate with Ras-transformed NIH3T3 cells and performed the same experiment that was done to find the cell density of non-transformed NIH3T3 cells. Then the students received a 6-well plate containing parental and Ras-transformed cells that had been starved for 24 hours in 2mL of media with 0.5% calf serum. The parental cells occupied the top left and middle wells and the Ras-transformed cells occupied the bottom left and middle wells. The students added 400 µL of calf serum (20%) and incubated the wells for 20 minutes. The culture medium was removed and cold PBS was used to wash the wells. The plate was immediately put on ice and 250 µL of cell lysis buffer was added to each well. The cells were scraped and the supernatants were transferred to Eppendorf tubes and incubated on ice for 20 minutes and centrifuged at 12,000 rpm in a cold room for 20 minutes. The supernatants were transferred to new Eppendorf tubes and put in a -20° C freezer. An SDS-PAGE and western blot were done for these cells in the same way they were done for the non-transformed cells (Dong).
Results:
The last page of the lab report contains the data from the experiment. Figure 1.1 shows the cell density of NIH3T3 cells. A hemacytometer was used to count these cells. The four numbers were averaged and multiplied by 2 in order to compensate for the dilution factor. This number was then multiplied by 10,000 in order to convert the units to cells/mL. These cells grew for one week before counting. Figure 1.2 shows the relationship between the concentration of the serum and the number of cells. As the results indicate, the cell density increases as the concentration of FBS increases. The cell density more than tripled when the FBS concentration increased from 2% to 10%. The cells were able to proliferate more in higher FBS concentrations because they received more nutrients that allowed them to survive and grow. Figure 1.3 shows the results from the SRB assay that was used to determine cell proliferation of the NIH3T3 cells. The SRB measures the absorbance of the cells, which can be used to determine the protein concentration. These results yielded similar results to the trend of increasing FBS concentrations. Furthermore, since these cells were given more time to proliferate their density was higher. Figure 1.4 shows the number of Ras-Transformed cells in each of the different concentrations of the serum. These numbers are extremely similar, which shows that the Ras-transformed cells are not dependent on the nutrients provided from the serum. This shows that the Ras-transformed cell density is independent of the concentration of serums or growth factors. Figure 1.5 shows the results from the SRB assay that was used to determine cell proliferation of the Ras-Transformed cells. This assay was similar to the one performed in Figure 1.3, the only difference was the type of cells examined. These results further proved that the Ras-Transformed cells grow independently of growth factors and nutrients because the absorbance and protein concentrations exhibited minimal change when the concentrations of FBS were changed. Figure 1.6 shows the western blot analysis performed on NIH3T3 fibroblast cells. This western blot was performed to examine the Erk ½ pathway of NIH3T3 cells without serum or growth factors, with calf serum, and with FGF. The control is lane 4, the cells with calf serum is lane 5, and the cells with FGF are in lane 6. This shows that the concentration increases greatly upon the addition of calf serum since that line is much darker than the control. The concentration should have also increased upon addition of the FGF, however it is evident that it did not. This is most likely due to the fact that the tubes containing FGF were old and the growth factors were no longer active. Figure 1.7 shows the western blot analysis performed on Ras-Transformed cells. This western blot was performed to examine the Erk ½ pathway of the Ras-Transformed cells without serum or growth factors, with calf serum, and with FGF. As is evident from the image, the addition of these substances that had increased proliferation in the non-transformed cells had little to no effect on the Ras-Transformed cells. This further proves that transformed cells are not reliant on the calf serum or FGF.
Discussion:
The purpose of this lab was to analyze the growth factor dependence of normal NIH3T3 cells and Ras-Transformed cells.
The goal of this experiment was to use cell cultures to monito the growth of these cells when treated with different calf serum and FGF concentrations. SRB protein assay was conducted in order to observe the cell proliferation. Western blotting was used to look at the relationship between the serum and activation of the Erk ½ pathway along with protein concentration. Thus, there were two hypotheses being tested during this experiment. This first hypothesis states: If various concentrations of Fetal Bovine Serum were added to non-transformed and transformed cells, the non-transformed cell density would increase with the concentrations of these substances and the transformed cells would be unaffected. The second hypothesis being tested is that the protein concentration of the Ras-transformed cells should also remain unaffected by the addition of serums and growth factors. However, the non-transformed cells will again increase with the concentrations of these
substances. The results obtained from this experiment support these hypotheses. The cell density of NIH3T3 increased greatly as the concentration of the calf serum increased. The density of Ras-Transformed cells, however, were unaffected by the addition of calf serum. This is because transformed cells are not reliant on nutrients to divide. The western blot looked at the protein concentration at different concentrations of the calf serum and FGF. It was concluded that the serum did affect the Erk ½ activation of the NIH3T3 cells. This is evident because the protein concentrations of the treated cells was higher than that of the control group, with the exception of FGF because it was possibly inactive. The Ras-Transformed cells, however, were not affected by these factors. Thus, the protein concentration remained constant as seen from the western blot. Since the serum did not affect the protein concentration, it is evident that the calf serum and FGF had no effect on the Erk ½ activation in the transformed cells. These results were expected because the non-transformed NIH3T3 cells have a finite life span and are dependent on calf serum for growth and survival. These cells grow by means of density-dependence inhibition, serum dependence, and/or anchorage dependence. The Ras-Transformed cells do no grow by these means and are able to proliferate infinitely independently of calf serum and growth factors. These results support the hypothesis. This experiment is significant to today’s society because of the rising cases of cancer. Cancer has affect nearly every person in some war. This experiment helps us to understand the difference between normal cell and transformed (cancer) cells. The growth of cancer cells can be analyzed by looking at the Erk ½ phosphorylation. This will provide an explanation of why and how cancer cells can grow at an increased rate. This knowledge can potentially provide new treatments and possibly a cure for cancer. A few potential problems with this lab could have been either human or experimental error. A human error could have been not incubating the cells for a long enough period of time after addition of trypsin. This would have resulted in some cells being attached to the plate and the overall cell count being lower than expected. The cell count could also be affected when counting the cells with the hemacytometer since the human eye is not fool-proof and may not have seen all of the living cells under the microscope. Another human error could be that the plate was not immediately put on ice after washing with PBS. This would also negatively alter the results. Furthermore, had some of the sample been lost when loading the wells of the gel, the western blot results could be altered. Aside from human error, experimental error could have also altered the results. For example, the FGF tubes that were used for the western blot analysis of non-transformed cells were old. This could be the reason why the sample with FGF was not visible on the western blot. If the FGF was inactive because of the age of the tube, it would not have induced growth of the cells like it should have. Furthermore, while transferring the samples and cells with the micropipette some of the cells may have remained in the tip. This would have lowered the cell count since solution would have been lost. Also, if the lab hood ventilation was not working properly that cultures would have been contaminated from outside air. In order to make this experiment more effective, an alternative approach could be used. Different types of cells could possibly be used. If the experiment involved epithelial cells, HeLa cells for example, the experimenter could compare the results from the different types of cells. Another alternative method could be the use of fluorescence instead of the western blot