Does glutamine in hybridoma cell culture medium need to be supplemented additionally?

For small-scale testing, set up controls: basal medium without supplementation, fresh basal medium, basal medium + 2 mM GlutaMAX, and basal medium + 2 mM alanyl-glutamine. Focus on observing cell viability, monoclonal antibody concentration, and light chain/heavy chain expression trends at 48 to 72 hours after inoculation. Do not test only one round; repeat at least two batches, because hybridoma clone secretion fluctuates significantly. If antibody secretion recovers after supplementation, it is recommended to aliquot the subsequent medium into single-use amounts after opening to reduce repeated opening, and store at 4°C protected from light; the 20 mL system can also be directly prepared into small packages of complete medium containing stable glutamine for convenient daily use and lot number traceability.

Glutamine in liquid basal medium does indeed degrade slowly during long-term storage at 4°C. The longer it has been opened and the more times it has been repeatedly warmed, the lower its actual concentration becomes. Given your later decline in antibody secretion, it is worth running a comparison experiment first: using the same batch of hybridoma cells, culture one group in fresh basal medium and another in old medium that has been stored for 3 to 4 weeks, then measure antibody titer and viability at 72 hours. If the difference is significant, supplement accordingly. GlutaMAX or alanyl-glutamine is more stable than regular L-glutamine. For a 20 mL small-scale test, you can first supplement to a final concentration of 2 mM and add it at the time of inoculation.

The 20 mL system is small, so supplementation must be accurate. Stabilized glutamine such as GlutaMAX is generally used at a 1× final concentration; for the specific dilution factor, refer to the instructions on the bottle. For alanyl-glutamine dipeptide, I usually set up three gradient groups at 1, 2, and 4 mM, with three replicate wells per group, and culture for 72 hours to measure antibody secretion. As for the timing of supplementation, it is recommended to add it at the time of inoculation, because the dipeptide form is relatively stable during the culture period and usually does not require repeated supplementation. If the pH drops rapidly in the later stage after supplementation, pay attention to using HEPES or adjusting the CO2 in the incubator to avoid pH fluctuations interfering with the results.

Check whether the basal medium formulation contains a GlutaMAX-type substitute. If it does, no supplementation is needed; if not, supplement a small amount.

Stable glutamine is better than regular glutamine, as it does not require frequent supplementation during prolonged culture.