Dose stimulated human PBMC with CD3, CD28 produce IL-4?
I did even kinetic measurements for IL-4 at 24 h, 48 h, 120 h but the cells did not produce IL-4!
dose any one have any suggestio???
A few suggestions to consider and troubleshoot the issue:
1. Check the functionality of CD3 and CD28 antibodies: Ensure that the antibodies used for stimulation are functional and capable of activating the T cells. You can test this by confirming their ability to induce other activation markers or cytokine production, such as IFN-gamma or IL-2.
2. Verify the viability and quality of PBMCs: Make sure that the PBMCs used in the experiment are viable and of good quality. Perform trypan blue exclusion or a viability assay to determine the viability of the cells. Poor cell viability can lead to reduced or altered cytokine production.
3. Optimize the stimulation conditions: Experiment with different concentrations of CD3 and CD28 antibodies and optimize the stimulation conditions. It is possible that the concentration used in the experiment is not sufficient to induce IL-4 production. Try a range of antibody concentrations and incubation times to find the optimal conditions for IL-4 production.
4. Consider the timing of IL-4 measurement: IL-4 production may peak at a time point different from the ones you tested (24 h, 48 h, 120 h). It is recommended to perform additional time points to capture the potential peak of IL-4 production. Try time points such as 6 h, 12 h, or 72 h to see if IL-4 production occurs earlier or later.
5. Evaluate other cytokines: If IL-4 production is not observed, it may be worth investigating the production of other cytokines, such as IFN-gamma or IL-2. This can help determine if the lack of IL-4 production is specific or if there is a general impairment in cytokine production.
6. Consider the donor-to-donor variability: It is important to note that PBMCs can exhibit donor-to-donor variability in terms of cytokine production. If the experiments were performed using PBMCs from a single donor, consider repeating the experiment with PBMCs from other donors to confirm the lack of IL-4 production.
7. Consider additional cell subsets: IL-4 production can be mediated by different cell subsets, including T helper type 2 (Th2) cells and mast cells. If IL-4 production is still not observed in PBMCs, it may be worth investigating these specific cell subsets separately.
Hope it helps:credit AI
Thanks Mohammed Immam In my hands, activated PBMC produce IL-5, IFN-g, IL-2,
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