I am working on T and B-lymphoma, and B16F1 melanoma cell lines. I would like to know if 2 uL is enough for 500 mL RPMI/DMEM +10% FCS+ 5.5mL antibiotic media culture system. Is it sufficient to control the oxidation pool ?
Regards
Bikash
Assuming that the stock (14.3 M) of beta-mercaptoethanol is fresh (and stored appropriately), addition of 1.9 microliter volume to 500 ml medium would give you the desired working concentration of ~55 micromolar. However, this concentration would change upon storage of the medium. Therefore, the best practical thing would be to prepare fresh medium and supplement it with beta- mercaptoethanol before adding medium to cells.
I did a search on SciGine and found the attached method for cell culture of B16F1. It seems like 0.055 mM is an appropriate mercaptoethanol concentration.
http://www.scigine.com/method.php?ID=104123
Assuming that the stock (14.3 M) of beta-mercaptoethanol is fresh (and stored appropriately), addition of 1.9 microliter volume to 500 ml medium would give you the desired working concentration of ~55 micromolar. However, this concentration would change upon storage of the medium. Therefore, the best practical thing would be to prepare fresh medium and supplement it with beta- mercaptoethanol before adding medium to cells.
Dear Professor Dr. Choubey,
Thank you for your valuable suggestions.
I make myself sure that, I am adding the exact concentration you have mentioned.
Sincerely yours,
Bikash
We grew several sublines of B16 melanoma cells over many years and never used any mercaptoethanol.
To get them growing fast (if that's what you want), the main thing is to plate at a low density, and use a lower pH of 6.9 to 7.0 (as with RPMI or supplemented MEM medium, 5-10% FCS and 10% CO2). We plated at 1 x 104 cells/ml, twice a week, and the cells would multiply by ~20 in 3 days or by ~60 in 4 days.
Dear Professor Dr. Bennett,
Thank for this information. My cell growth is good in DMEM. I was worried if without mercaptoethanol the ROS can affect the growth. With your valuable message and work experience in B16-F1, I could imagine that I don't need mercaptoethanol as a mandatory chemical for the culture media. I will try to monitor the cell growth with and without mercaptoethanol.
Sincerely yours
Bikash
The survival of lymphocytes, lymphoma and melanoma cells can be enhanced by 2-mercaptoethanol. These cells can not use the cystine in the cell culture very easily but adding 2-mercaptoetanol makes this possible.
Please check the attached file.
We also use 2-mercaptoethanol in RPMI1640. The final concentration is 55 uM.
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