Hello all, I am using S2 cell line to transfect a pMT construct to check the expression pattern. After induction of expression with CuSO4 addition my cultures became yellow after 12 hours and i did not get any expression of the desired protein. Is it because of bacterial infection? If so, why it did not happen during transfection? Any suggestion will help a lot. Thanks
It could be that contamination was introduced with cuso4. Did you filter it? Since s2 grow in suspension it is hard to see if there is bacterial infection, except plating bit of media on a lb plate.
The other reason for yellow media is change of pH, which could have an effect on expression of your construct.
Hope this helps
Cell culture or the medium which is turned yellow.? If it is the medium, it may be due to bacterial contamination. Did you get any protein other than the desired one..?
do you have phenol in your cell media? If that turned yellow it indicates that the pH has drastically changed. However, since the color changed very quickly, there could be some contamination as well. I would definitely check the pH.
Assuming the media contains phenol red and the pH has NOT shift due to the accumulation of acidic metabolic byproducts, a likely cause is the indirect oxidation of phenol red. This can verified with a very simple test. Copper can catalyze lots of auto oxidations (e.g., those of thiols) leading to low steady state levels of hydrogen peroxide in the media. Hydrogen peroxide can then indirectly oxidize the phenol red to produce a yellow product, this oxidation is facilitated by peroxidase activities (either of cellular origin or from media components, or possibly even by copper chelates). This phenol red reaction is the basis of a hydrogen peroxide assay that was discovered by investigating the pH independent yellowing of tissue culture media under certain conditions.
Pick E, Keisari Y. A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture. J. Immunol. Meth. 1980; 38:161-170.
If you get some of the supernatant and raise the pH to ~14 with normal phenol red you’ll get a purple color, whereas the oxidized phenol red will give a more blue color. You can verify this by measuring the absorbance at 610 nm where a large increase will be observed if this is indeed the process occurring.
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