Hi all,
I am planning on performing a Geneticin kill curve procedure on HEK293 and SHSY5Y cells before I carry out my plasmid transfection with these cell lines. Thus, I would like to put forth my planned procedure here. Hopefully you can help me correct it if there are flaws.. I also have some questions, which I hope you can help clarify.
1. Seed 6000 cells per well in a 96 well plate in complete medium
Questions:
- I have been using DMEM with antibiotics and serum, but I have read that I should remove serum also. Should I do it? I will not be adding penicillin-streptomycin anyway
- Would 6000 cells per well be ok to achieve 50% confluence the next day? Would this cell number cause overconfluency over my 2 weeks of observation?
2. After the cells reach 50% confluence, replace medium with Opti-MEM and the appropriate range of antibiotics (I am planning on using 0 to 1000ug/mL of geneticin) and keep wells in triplicates. Replace with fresh medium every 2-3 days and visually observe the cells
Questions:
- I am not planning on doing an MTT assay till the 10th or 14th day. Would that be ok to just visually monitor over the duration until the very end?
- Is it ok to use Opti-MEM, as that will be the medium I will be using for my cells when transfecting with lipofectmain 2000?
Thank you so much and I hope you can help me! If anyone has done this before with HEK293 or SHSY5Y, kindly share your experiences too!
Best Regards,
Mathangi
hi..
It is generally recommended to remove serum from the culture medium when performing a geneticin kill curve procedure. Serum can contain growth factors that can affect the sensitivity of the cells to geneticin, so it is important to use a serum-free medium in order to obtain more accurate results. Opti-MEM is a good choice for a serum-free medium.
6000 cells per well should be sufficient to achieve 50% confluence the next day. However, it is important to monitor the cells carefully and adjust the cell density as needed to avoid overconfluency. Overconfluent cultures can produce unexpected results, so it is important to maintain the cells at the appropriate density.
It is generally recommended to use an MTT assay or other type of viability assay to quantitatively measure the sensitivity of the cells to geneticin. Visual observations can be subjective and may not provide as accurate a measure of cell viability. However, you can use visual observations as a supplement to a more quantitative assay if desired.
It is generally acceptable to use Opti-MEM as the culture medium for the geneticin kill curve procedure, as long as it is serum-free. However, it is important to make sure that the medium is compatible with the geneticin concentration that you are using. Some media may not be suitable for use with high concentrations of geneticin, so it is important to consult the manufacturer's recommendations or test the compatibility of the medium before using it.
I hope this information is helpful. If you have any further questions or concerns, please do not hesitate to ask.
Regards,
Mohammad Alzeyadi Hi! Thank you for the useful information:) This more or less clarifies whatever I would need to perform this procedure!😇😇😇
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