as you can see from the attached picture 1) how I can avoid cell aggregation. I have tried everything and still, it's frustrating because it affects my picture quality. What kind of neuron is N2a? Differentiation to which type?
2) for the protein expression, how can I locate it because it should bind so some ion channel
3) My labmate says that after the transfection under microscopy, they observe the protein expression moving( unlocalized). I did not face this. Is it possible?
4) i am planning to do a pH study to test how my protein is sensitive to pH change. I am planning to do this pH 6.6, 7, 7.2,7.3,7.4,7.5,7.6,7.7.7.8,8,8.5 what do you think? and how much should I wait after changing the pH for my cell to adjust to the new pH?
5) for preparing different solutions with different pH. Is it OK if I use my complete medium (which has FBS and antibiotics) and adjust its pH using NaOH and HCl? Is it OK or might it kill my cells?
6) as shown in the second picture, I think the GFP accumulates in the center. I believe is an uneven distribution. I may be wrong, so feel free to correct me. Also, after differentiation, I see that not only 1-2 cells that express GFP differentiated, and even if they do, it did not go to the axon. I was except that I will see some GFP on the axon because there are some ion channels.
Thank you
1) Avoid cell aggregation by using a cell culture medium that does not contain serum. Serum contains proteins that can promote cell aggregation. Add a chelating agent that binds to calcium ions. Proper handling, centrifugation, and anti- contamination techniques.
2)Locate with Western blotting; the protein of interest is separated from other proteins in a sample by electrophoresis. Other options are immunohistochemistry and fluorescence microscopy.
3) Yes, it is possible. The protein of interest is tagged with a fluorescent molecule, which allows it to be visualized under a fluorescence microscope and it will move within the cell.
4) Sounds good. You should wait at least 30 minutes after changing the pH for your cells to adjust to the new pH.
5) It is not recommended to use NaOH or HCl to adjust the pH of cell culture medium that contains FBS and antibiotics. This is because these chemicals can denature proteins and other molecules in the medium, which can damage the cells.
6) The distribution of GFP can be uneven in cells. In some cells, GFP may accumulate in the center of the cell, while in other cells, it may be more evenly distributed.
John Odonnel thank so much
in here Yes, it is possible. The protein of interest is tagged with a fluorescent molecule, which allows it to be visualized under a fluorescence microscope and it will move within the cell. ( if it is moving does that mean unlocalized of the protein ?)
in here 5) It is not recommended to use NaOH or HCl to adjust the pH of cell culture medium that contains FBS and antibiotics. This is because these chemicals can denature proteins and other molecules in the medium, which can damage the cells. ( so what should I use to adjust the pH and which media)
in here The distribution of GFP can be uneven in cells. In some cells, GFP may accumulate in the center of the cell, while in other cells, it may be more evenly distributed. ( why is that unlocalized ?)
Proteins can move within cells without being unlocalized. Examples are diffusion, facilitated diffusion, active transport, exocytosis, and endocytosis.
If you need to adjust the pH of your cell culture medium, it is best to use a buffer solution. A buffer solution is a solution that resists changes in pH. You can find buffer solutions that are specifically designed for cell culture.
There are a number of reasons why the distribution of GFP can be uneven in cells. The expression level of GFP can also affect its distribution. GFP is often more evenly distributed in non-dividing cells than in dividing cells.The presence of other proteins can also affect the distribution of GFP. GFP may be more likely to accumulate in areas of the cell that are exposed to certain chemicals or conditions.
Thank you, I enjoyed being apart of your study.
Tris, MOPS, PIPES, or HEPES are good versatile buffers.
Cell aggregation can be avoided by increasing the culture medium with a non-toxic agent like serum-free media or polyethylene glycol. Additionally, providing adequate levels of growth factors and nutrients to the culture medium can also help reduce cell aggregation. N2a is a murine neuroblastoma cell line derived from a spontaneous tumor in a BALB/c mouse. It is a differentiated cell line, meaning it has already gone through a process of differentiation from a stem cell to a more specialized type of cell. N2a is a type of neuron, specifically a sympathetic neuron.
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