I am culturing NK92 now. Last week, I used up the ATCC recommended medium and I made a new one just the same way I did before. The way I made the medium is as follows:

1000ml αMEM （Gibco 12561）;

FBS 167ml(Atlanta Premium);

horse serum 167 (ATCC);

folic acid (sigma F8758) powder stored in RT, dissolve it in NaOH, then filtered and add directly to medium;

myo-inositol (sigma F8758) powder stored in RT, dissolved in PBS, then filtered and add directly to medium;

2-mercaptoethanol (M6250) stored in 4 ℃, taken from others and placed in 15 tube, instead of brown glass bottles as  original package;

il-2 200U/ml;

antibiotics.

I changed medium for cells right after I made the new medium;

The cells were growing very well originally, but after I change the newly made medium, most of them died over night. I don't know why because I just did the same way I made the medium.I thought reasons might be like this:

1. I didn't shake the medium so it's not well mixed, and the supplements are not equally distributed.

2. Something has degraded or turn to toxic after a long time. It most possibly be 2-mercaptoethanol because I borrowed it from others and stored in 4℃ for 4 month without avoiding light and the stored condition is not the same as the original package.

But I am not sure what exactly the problems can be.

Did any one can help me with the problems? Thank you.