For intracellular flow cytometry staining of IL-17A and IFN-γ, must the stimulant and blocking agent be from the same brand?

Mixing different brands generally does not directly cause stimulation failure; it is more a matter of matching the flow cytometry staining system. Stimulants and blockers are of chemical or microbial origin with well-defined mechanisms of action, whereas antibody clones, fluorochromes, and fixation/permeabilization buffers have a greater impact on results. It is recommended to use fixation/permeabilization reagents from the same brand as the antibodies to optimize intracellular staining; stimulants only need to have normal activity. Before a 20-tube experiment, run a 2-tube pilot to observe lymphocyte morphology and viability after stimulation. Storage after opening: PMA/ionomycin DMSO stock solutions at -20 or -80 protected from light; BFA at 4°C for short-term; avoid repeated freeze-thaw cycles.

I generally don't get hung up on brands, but I do look at the solvent and recommended dilution factor for BFA. BD GolgiPlug, BioLegend Brefeldin A, and Invitrogen are interchangeable, with a typical working concentration of 1x, used the same day after dilution; PMA/ionomycin are dissolved in DMSO and aliquoted to avoid repeated freeze-thaw cycles. If the background increases after mixing, it may be due to excessive BFA concentration or prolonged PMA stimulation, leading to cell death and nonspecific staining. It is recommended to perform a time gradient: 4, 5, and 6 hours, and select the time point where cell viability is >80% and the positive population is clearly separated. Unstimulated controls and isotype controls must be included; otherwise, it is impossible to distinguish true positive signal from background.

PMA, ionomycin, and BFA do not have to be the same brand as the flow cytometry antibodies. In our lab, BD antibodies have long been mixed with BioLegend's Brefeldin A and Sigma's PMA/ionomycin, with stable results. Differences between brands mainly lie in purity, dissolution method, and solvent (commonly DMSO). It is recommended to uniformly use anhydrous DMSO to prepare high-concentration stock solutions and aliquot them. However, if a system has already been optimized for time/concentration, it is best to perform a small-scale pilot experiment before mixing, comparing the percentage of IL-17A/IFN-γ-positive CD4+ T cells and cell viability. High background is mostly a matter of concentration or stimulation time, rather than brand compatibility.

It should be noted that different species/cells have different sensitivities to PMA and ionomycin. Mouse splenic lymphocytes typically require only 4-6 hours of stimulation, and PMA 50 ng/mL + ionomycin 1 μg/mL + BFA 1x is commonly used; if the cells are not in good condition, PMA can be reduced to 20-30 ng/mL. Ionomycin is easily damaged by repeated freeze-thaw cycles, so it is recommended to reconstitute it in DMSO after opening and aliquot it, with each tube containing a single-use amount. A small size is enough for 20 tubes, but if multiple experiments are planned, it is better to purchase a slightly larger size at once and aliquot it for greater stability.

For 20 tubes of samples, with the stimulation system calculated at 1 mL/tube, approximately 20-24 mL of working solution is required. The commonly used final concentration of PMA is 20-50 ng/mL, ionomycin is 1 μg/mL, and BFA is typically used at 1x. Small specifications such as PMA 0.1 mg, ionomycin 1 mg, and BFA 1000x small packs are usually sufficient, but the key point is not to repeatedly use the working solution after preparing a large volume at one time. It is recommended to aliquot the PMA/ionomycin stock solution at 5-10 μL/tube and store at -20°C protected from light; aliquot BFA according to single-use amounts to avoid repeated warming. After opening, refer to the instructions for shelf life; generally, DMSO stock solutions can be stored for several months, but activity will gradually decline.