Troubleshooting

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.

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