Hi,
I begin with MonoMac6 culture and I have lots of problem.
First, recommanded medium is RPMI 1640 supplmented with L-Glutamine, 1mM sodium Pyruvate, 9 mM bovine Insulin 10%to 20% FCS, and maybe Oxaloacetic acid. But no paper, no data sheet, no publication say if this medium should be balanced with HEPES or Sodium bicarbonate, and which is the culture pH.
I tried to culture these cells in RPMI 1640 with all things, but not balanced, and as anyone could realize, once @ 37°C, cells die. So I tried to culture them in DMEM-F12 10 %FCS, balanced with HEPES and Sodium bicarbonate, once again, cells die. Cells are cultured in 24 well falt bottomed culture plates, as recommanded.
I don't know how proceed to rescue the little amount of cell I've left.
Does anyone culture these cells and know any potential responses dealing with my matters?
Thanks.
0
1 reply to this topic
#2
KDHughes
-
- Active Members
-
- 7 posts
member
0
Neutral
Neutral
Posted 22 May 2003 - 07:59 AM
I haven't used that particular cell line, but I've used that medium with other cell lines.
In everything I can find for RPMI 1640, the medium can be buffered with HEPES, Na bicarbonate, or both. Use 2.0 g/L for bicarbonate, and 5.958 g/L of HEPES. In all cases pH should be adjusted to a range your cells can stand. It varies from one cell line to another. I've had pretty good luck at pH 7.1, even with cell lines that like a slightly more alkaline environment. Remember too that media pH can drift with storage. To keep a valuable and touchy cell line happy, check the media pH every time you use it. You might also consider gassing the headspace in the media bottle with 5% CO2 before you put it away.
I don't typically use insulin in media containing serum, but when I do use it, it's at 5 mg/ml. I think that calculates to roughly 1 mM. Check to make sure your insulin dissolves - I make a stock solution in water at pH < 3. The stock can be frozen for use later.
I would keep the amount of serum in the medium below 20%. Some cells do exhibit serum toxicity, so that levels that high actually kill them off. Try to baby your cells along at 10%. Glutamine levels for RPMI are specified at 2 mM, but I've had success bringing back wounded cells with a slight boost in glutamine levels - so you might try using 4 mM.
Last thing - pH is absolutely vital to most cells, and with RPMI I've used both 5% and 10% CO2 to gas the cultures. Check what CO2 environment you have your cells in. Good luck, and feel free to write to me directly if you need more clarification.
In everything I can find for RPMI 1640, the medium can be buffered with HEPES, Na bicarbonate, or both. Use 2.0 g/L for bicarbonate, and 5.958 g/L of HEPES. In all cases pH should be adjusted to a range your cells can stand. It varies from one cell line to another. I've had pretty good luck at pH 7.1, even with cell lines that like a slightly more alkaline environment. Remember too that media pH can drift with storage. To keep a valuable and touchy cell line happy, check the media pH every time you use it. You might also consider gassing the headspace in the media bottle with 5% CO2 before you put it away.
I don't typically use insulin in media containing serum, but when I do use it, it's at 5 mg/ml. I think that calculates to roughly 1 mM. Check to make sure your insulin dissolves - I make a stock solution in water at pH < 3. The stock can be frozen for use later.
I would keep the amount of serum in the medium below 20%. Some cells do exhibit serum toxicity, so that levels that high actually kill them off. Try to baby your cells along at 10%. Glutamine levels for RPMI are specified at 2 mM, but I've had success bringing back wounded cells with a slight boost in glutamine levels - so you might try using 4 mM.
Last thing - pH is absolutely vital to most cells, and with RPMI I've used both 5% and 10% CO2 to gas the cultures. Check what CO2 environment you have your cells in. Good luck, and feel free to write to me directly if you need more clarification.
