What is the effective concentration of recombinant human IL-2 to stimulate T cell proliferation?

Batch stability I mainly look at three points: biochemical, endotoxin, and sterility in COA. Insufficient domestic activity is sometimes related to the freeze-drying process and storage conditions. It is recommended to pack the goods in small volumes and keep them away from light at -20 °C or -80 °C to avoid repeated freezing and thawing. After dilution, do not leave the working fluid for a long time. I am used to using PBS or basic culture medium containing 0.1% BSA, and it is used up on the same day. When comparing suppliers, ask if the other party can provide bioactivity test data, such as CTLL-2 cell proliferation ED50 or similar methods, and then look at the consistency between the batches. If the small package is tested first, buy the same 10 μg or 50 μg, and do the proliferation promotion curve in parallel with the brand you are using now, focusing on EC50 and the maximum proliferation multiple. The price should not only look at the unit price, but should be spread to each well or per ml according to the actual active unit. Domestic production may be cheap but the price is low. Imported small packages with cold chain delivery periods usually need to ask if they are in stock. If the low-temperature transport is not in place, the activity will be reduced, and the temperature record will be confirmed when the goods arrive.

For the quantity of 10 ^ 7 primary T cells, I suggest calculating the total activity requirement first. For example, in a 100 IU/mL or 10 mL system, 1000 IU is required per day; if the IL-2 specific activity is around 10 ^ 7 IU/mg, a 10 μ g small package for one round of gradient screening is usually sufficient. Attention should be paid to the activation process. IL-2 itself cannot replace the first signal, and most primary T amplifications still require anti-CD3/CD28 magnetic beads or coated antibodies to be activated first. IL-2 is mainly used to maintain proliferation and survival. In terms of stimulation time, it is common to supplement IL-2 about 24 hours after activation, and then change the medium by half according to the color of the culture medium and cell density, and continue to supplement until the amplification is complete. In addition to proliferation, it is recommended to add CD25 and phosphorylated STAT5 as detection indicators, which are more meaningful after 72 hours of observation. If significant differences are found between different batches, the same batch of cells can be divided into several groups, with fixed activation conditions, and only IL-2 batches can be used for comparison. In terms of supply, domestic spot goods are generally fast, and for imported small packaging, you need to inquire about the cold chain delivery time. Don't wait until the last week to buy.

When expanding primary T cells, I usually set the working concentration of IL-2 at 50-200 IU/mL. If we calculate by quality, different brands have different specific activities. I suggest starting with three gradients of 10, 50, and 100 IU/mL. Start supplementing 24-48 hours after activation, change the amount of fluid every 2-3 days and replenish IL-2. If 10 ^ 7 cells are cultured at a rate of 10 ^ 6/mL, approximately 10 mL of the system is required. The amount of supplementation is not large, but it needs to be continued until the 7th to 10th day. I usually check the activation of CD25/CD69 within 72 hours, and on the 5th day, I use CFSE or EdU to observe proliferation while recording the activity rate. Sometimes the insufficient activity in domestic products is not due to insufficient concentration, but rather a difference between the labeled activity and the actual potency. It is recommended to use your own target cells to create a dose curve after arrival, rather than just diluting according to the instructions. Small packages are generally 10 μ g or 50 μ g, and there is a significant difference in price and delivery time between imported and domestic products. For imported cold chain small packages, it often takes 1-2 weeks to contact agents, while domestic products have more stock available.