Help with MonoMac6 - culture problem (May/21/2003 )

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.

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.