I'm using Leibovitz L-15 medium complemented with 20% FBS, 2mM L-Glutamine, 1mM sodium pyruvate,
Does adding 2-ME (mercaptoethanol) help freshen the cells?
please check the links
www.ncbi.nlm.nih.gov › NCBI › Literature › PubMed Central (PMC)
https://www.corning.com/media/worldwide/cls/documents/cc_scale_up_guide.pdf
www.gelifesciences.com/file_source/GELS/.../Microcarrier%20Cell%20Culture.pd
www.vanderbilt.edu/viibre/CellCultureBasicsEU.pdf
https://books.google.co.in/books?isbn=0125441614
Dear Nazanin,
I am not sure what you mean by "freshen" the cells, and you do not mention what kind of fish cells you are trying to culture. I have never used 2-ME my culture of adult fish retinal ganglion cells. However, you might take a look at this RG discussion:
https://www.researchgate.net/post/Has_anyone_used_2-mercaptoethanol_in_their_cell_cultures/1
Good Luck!
Jill
Dear Jill,
thank you for your useful comment. I'm working on sturgeon fish. It seems that cells are very unstable in the culture medium. they die after a week. I tried to add 2-ME to the media to reduce ROS but actually that didn't help.
I'm now trying some other factors. but I could still use your help. What do think the problem can be?
Nazanin
Dear Nazanin,
I can't say for sure what your problem may be. Primary cells don't last forever, but dying after only one week is pretty fast. How often do you change your media? I usually plan on every 2 or 3 days, and I change by 1/2 to 2/3. But you may need to change your media on a different schedule, depending on your initial cell density. The L-15 has phenol red in order to monitor the pH, just like other cell culture media, so you can tell if your media is too basic or acidic and needs to be changed. Depending on your cell type's needs, you may find you get better pH control with the addition of HEPES buffer. I believe you can get commercially available L-15 with either 15 or 20 mM HEPES already added.
My suggestion would be to start with the same conditions used in mammalian cells (or even frog or axolytl cells) of the same type that you are trying to culture (i.e., corneal, epithelial, muscle, fibroblasts, or whatever). The only exception with the L-15 is it buffered for room air levels of CO2 (not 5%). I simply culture fish neurons at room temperature, but in the dark. Because it is easy to use room air, we made our own incubators out or polystyrene containers with loose fitting lids, and spray-painted the OUTSIDE of the containers with the black spray paint made for plastic that is available here in the US. We put a plastic support grid in the bottom of the container to hold the dishes, and to allow us to put sterile towels saturated with sterile water underneath the dishes in order to provide humidity. Even though they are dark boxes, I still keep them in a cupboard, or in a room that is usually dark.
Good Luck!
Jill
Dear Jil,
thanks for the great information. Do you think that darkness is necessary for fish cell culture or is it for the neurons? we have the cool incubator for fish cell culture which is practically a fridge and the glass door is covered with aluminium foil. So it is dark.
By the way, I tried hydrochortisone which seems to be helpful. The cells are fresh and hopefully proliferating.
I was planning to add EGF, too but we only have access to human EGF! do you think it could work on fish cells too??
Thank you in advance
Nazanin
Dear Nazanin,
I am glad the hydrocortisone seems to be working. I only have experience with such supplements in serum-free media formulations.
In general, most cells need to be cultured in the dark. Phototoxicity is also why one must be careful how much light is used for time lapse phase microscopy, even if you have installed an infrared filter to block the heat from the lamp. Also, amino acids (and other culture media components) are light sensitive. That is why one should store media in a dark fridge.
Speaking of refrigerators, might it be possible that your incubator is TOO cold? It is very difficult to culture poikilotherm animal cells well if it is too far below 60-65F (18-20 C?).
As for supplements, I have always used whatever was commercially available for rodents for the fish. I would like to point out that investigators that induce frogs or fish to spawn use human HCG. So, human EGF should be fine. If I can't find any info on PubMed for publications listing a different media supplement concentration, then I usually use the same as recommended for the mammalian cells.
Good Luck!
Jill
Dear Jill,
Thanks for your great help
I'll try EGF for sure. I'll let you know about the results.
thanks
Nazanin
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