Do you want to switch to phenol free red DMEM for fluorescence imaging? Which brand is more stable?

High fluorescence background is not necessarily caused by phenol red alone. It is recommended to first run three parallel conditions: fresh phenol red-free medium, old medium imaged in PBS, and imaging buffer background. MCF-7 cells themselves have autofluorescence, and the plate bottom and serum lot can also contribute to background. Some people see significant improvement after switching to phenol red-free medium, but others find that the source is phenol red/riboflavin in the serum. After selecting phenol red-free medium, perform a signal-to-noise ratio test and compare background values under the same probe concentration. If the background is not reduced sufficiently, also check the filter channel and staining concentration.

The pH of phenol red free culture medium cannot be determined by color alone. After opening the bottle, it is more reliable to use a pH meter or take a small amount of sample for reading. It is recommended to pack the goods into 50 mL/tube on the ultra clean bench after arrival, store them at 4 ° C in the dark, and avoid opening large bottles repeatedly. After opening the bottle, if the operation is standardized, I usually use it within 4-6 weeks. If it exceeds, I will first confirm the clarity and pH, and will no longer use it for critical imaging. MCF-7 is sensitive to medium replacement. Before replacing with phenol red, perform fluorescence background comparison and growth curve, and do not suddenly switch in the same batch of experiments.

Phenol free red high sugar is usually not unreasonably expensive than regular high sugar, but the import lead time may be longer, and both Gibco and Sigma have out of stock cycles. Suggest asking the agent to report whether 'spot goods can be shipped' or 'overseas transfer of goods', and to provide a written estimate of the expected arrival time. Don't just look at the catalog price when it comes to pricing, as there may be a significant difference after including tax and shipping. If the laboratory is not always available, 2-3 bottles can be ordered at once, and after stable testing, small batches can be stocked; The shelf life of phenol free red after opening the bottle is similar to that of regular DMEM, so there is no need to stock up too much at once.

I have used both Gibco and Sigma's phenol free red high sugar DMEM, and the difference in stability is not as significant as I imagined. The key is not to mix versions containing HEPES. If you are in a HEPES free system, the pH will quickly rise after leaving the incubator, and the purple color may not be a brand issue, but rather due to prolonged exposure to air. Gibco liquid has good consistency between batches, while Sigma with the same specifications usually has simpler packaging but clearer ingredients. You can ask the agent to compare the COA of two companies with the concentration of sodium bicarbonate. Reduce the number of lid openings during live cell imaging and try to pre balance the culture medium before imaging.