The purpose of this project is to figure out whether or not if PNUTS can be knocked down and see an increase in apoptosis will occur in cell lines MCF7 compared with MCF10A which are both grown in 3D cell cultures. After Trouble shooting for almost two months we have received very promising results. The red Stain, which is called Ethidium Homodimer, and the green fluorescent stain is called Calcein AM was used from the Live/Dead kit to analyze cell death. We saw that in the cell line MCF7 (cancerous cell line) when transfected with a RNA strand there were more dead cells which were stained bright red (an indicator that the cell died) then the cells that were not transfected with RNA (control group). We saw that the control group was stained brighter fluorescent green, which is a good indicator that many of the cells were alive and healthy. These results showed us that not only did the Live/Dead kit apoptosis analyzer work, but that cancerous cells in 3D shape when treated with drug have higher death numbers then the cancerous drugs that are not treated with drug. In the MCF10A cells when we stained them with the live/Dead kit it gave us great images of their morphology. The MCF10A when grown in a 3D shape are suppose to take the shape of an Acini, which is a circle of cells with no cells in the middle. Our results showed us that the outer cells were alive and bright green, but the cells in the middle were dead bright red. We are thinking that the dead cells in the middle that are stained red are the ones that die in order to create this Acini shape. This is something we will investigate in future experiments. We also saw a fair amount of equal live cells in the MCF10A control group and drug group which is what we predicted would happen being that MCF10A cells are normal and not cancerous. We would like to continue our experiments to make this project publishable in the near future, but so fair our results have been very promising.
This project has taught me many skills such as independence, maturity, responsibility, and many other skills. I learned how to do certain math in order to figure out how much volume to use of substance depending on the cell type. I learned how to talk about my research fluently in a language that is understandable to individuals that have little knowledge in this field. I am able to give presentations on my research and write reports on it, which makes me happy because when I first started this project I was unable to do this. I see a big improvement in my skills and really love being in the Undergraduate Research program. Thank you.
Professor Krucher and I have continued troubleshooting the apoptosis marker which is using Active Caspase 3, which stains the cells that are going through apoptosis fluorescent green. This is the procedure that will allow us to look and see cells that are undergoing cell death, apoptosis. We are only using MCF7 cells for this process being that they are the cancer cell line. We are continuing to use the roscovitine drug in order to induce apoptosis. We are also performing these trouble shoot tests in 2D and 3D cells cultures in order to make sure that it can be done in both and also see if the 2D will give a clearer picture. We also just purchased a Live/Dead kit which is also a apoptosis marker and works in the similar fashion as the Caspase 3 marker. We would like to try other techniques to receive better quality, easier, cheaper, and less time consuming way of staining. We are also trouble shooting another way to extract the protein from the 8 welled slides containing the 3D cells being that when we need to do western blots we get a clearer band when we’re looking for specific proteins like RB (Retinoblastoma Tumor Suppressor). Overall, we’ve been successful with each step towards our overall goal besides the apoptosis marker coming out more defined even though it was successful. We now would like to put the steps together. Kind of like a puzzle piece in a way. Each piece was successful, but now we have to put it together which will hopefully lead to a successful knockdown of PNUTS in breast cell MCF7 and be able to compare it to MCF10A’s.
Learning experiences and challenges: Challenges so fair would be that due to the room temperature in the lab, some of our 3D cell cultures grew in a 2 dimensional form because the matrigel solidified way to fast while plating. We placed our slides on ice baths to hopefully avoid this in the future, which leads to saving our cells for further observation. I also have a hard time with conversions which is needed while troubleshooting the apoptosis marker kits. I continually learn how to convert volumes in order to obtain what I need for the experiment or tests that are required. I am also learned through the articles how to maintain different cells types that are cancerous which may be used in our future experiments alongside with the MCF7. That’s all for now. Thanks for reading.
Recently Professor Dr. Krucher and I have successfully extracted the molecules from the matrigel of the 3D wells and were able to identify them. In other words, both cell lines are being grown in small welled plates in a matrigel which is what causes them to grow into a 3-dimensional structure. We were able to take the cells out of the matrigel to specifically look for certain molecules such as RB (Retinoblastoma Tumor Suppressor) and Rb phosphorylation site 821. This is important because in the long term we plan to activate these molecules in order to cause cell death, so we have to be able to detect them. Our next direction we would like to go is to test different methods to measure, analyze, and observe cell death with these cell lines being in matrigel. We have not yet come up with a protocol, but are working on ways to be able to treat and analyze the cells so we can quantify cell death in order to see if cells do respond to drugs in a similar manner as when they would in a 2 dimensional structure.
Personally, I think these results are very good because we were worried that we would not be able to extract the cells from the matrigel in order to target certain molecules. We were also worried that these cell lines would not grow in a 3 dimensional structure, but we were able to see using a high tech. microscope that there is evidence of them growing the way we had hoped. Being that it takes at least 16 days for these cells to grow up in a healthy state to be used for experiments; our findings are spread out over a course of a few weeks and are coming along slowly. We also had a big set back after hurricane sandy and new cells had to be grown. We had to push back our dates for trying out the cell death signaling protocols and so there will be more on those findings in my next blog.
Thank you for Reading.
There is much that goes on while a cell divides. There are check points a cell must go through in order to move forward and steps that must happen in order for the cell want to divide. In cancer cells, certain steps are either mutated or knocked down which causes the cell to uncontrollably divide. We would like to focus on what major steps or activities that would allow for the cell to want to committee suicide. Knocking down PNUTS which is a protein that is necessary in order for the cell to proceed to division has shown when knocked down levels of cell death increase in 2D cell cultures.
The purpose of this project is to figure out whether or not if PNUTS can be knocked down and see an increase in apoptosis will occur in cell lines MCF7 and MCF10A that are grown in 3D cell cultures. An increase in apoptosis has already been observed in cell lines with PNUTS knocked down while being grown in 2D cell plates, but we want to say if it stays true in 3D plates because its more physiologically closer to our bodies. The goals are to knock down PNUTS in these cell as well as to learn how to analysis levels of apoptosis. I expect to learn many things from this project because growing cells in 3D is new to our lab. I would like to learn how to analysis apoptosis and proteins in these cultures. The methods we will use for our research would be to use wells to host the matrigel and cells to grow. We would like to grow the cells in culture for at least 16 days and add drugs ( Roscovitine, 5′ strand where we insert RNA into the cell, etc) to see if there is an increase of apoptosis.