For culturing HepG2 and RAW264.7, should DMEM high glucose or 1640 be used? Is glutamine supplementation needed?

For small-scale laboratory use, I recommend purchasing a uniform high-glucose DMEM containing stable glutamine, which works for both HepG2 and RAW264.7 cells and simplifies inventory. Sodium pyruvate is not essential for these two cell types; if the medium lacks it and you perform high-density or long-term culture, you can add it at 1 mM, but do not do so blindly. Upon receiving a new batch, aliquot it first and conduct small-scale testing: seed one flask each of the two cell types, observe adherence morphology and doubling over 72 h, and record pH changes. Do not hesitate to replace basal medium that has been opened for more than 4 weeks—just switch to a new one. Keep a small bottle of GlutaMAX 100× stored at 4°C and supplement as needed.

If 1640 medium becomes alkaline after being opened for one month, first check whether the bottle cap is sealed and whether the refrigerator temperature fluctuates greatly. It is recommended to aliquot new medium directly into 50 mL centrifuge tubes upon arrival, with each tube sufficient for 2-3 uses, to reduce the air headspace. Normal fresh 1640/DMEM is orange-red to peach-red; a pinkish-purple color indicates over-alkalinity, while a yellowish color indicates over-acidity. If slightly alkaline, the cap can be loosened in a biosafety cabinet and the medium returned to a 5% CO2 incubator to equilibrate for a while before use, but repeated pH adjustment is not recommended. If the color is obviously abnormal or turbidity is already present, do not risk it—discard it directly.

In commercially ready-to-use formulations, L-glutamine degrades slowly at 4°C, typically showing a significant decline starting 3-4 weeks after opening, so the deterioration in cell condition after one month of using 1640 may be related to this. Currently, many basal media use GlutaMAX or alanyl-glutamine as substitutes, which offer much better stability and are less prone to ammonia production at 37°C. If you are using the regular L-glutamine version and it has been more than 3-4 weeks since opening, it is recommended to supplement with GlutaMAX to approximately 2 mM, or supplement according to the original formulation, but do not add twice. After supplementation, the medium can be filtered through a 0.22 μm filter and aliquoted to reduce contamination.

For HepG2, I typically use high-glucose DMEM (4.5 g/L glucose) supplemented with 10% FBS, and the growth and morphology are relatively stable; RAW264.7 can also be cultured perfectly well in high-glucose DMEM, and some labs use RPMI1640 as well, but 1640 has a different buffering system and nutritional emphasis, and if it is not stored properly after opening, it can indeed tend to become alkaline. If you want to standardize both cell types, you can simply use high-glucose DMEM for both, reducing inventory and cross-contamination risks. Adjust the medium change frequency according to density; once RAW264.7 reaches higher density, it acidifies the medium relatively quickly, so pay attention to the color. Do not switch frequently between DMEM and 1640, as the cells need to adapt.