We noticed that macrophages from anti-CD40 injected mice, after we sort them, became very activated so that they don’t need another stimuli (TLR4 or TLR9 agonists) to produce high levels of nitric oxide in vitro. Does someone know if sorting procedure can trigger (aggravate) already primed macrophages? References? Thank you in advance.
Dear Alexander, one of the explanation that sorting itself led to temporary oxidative stress (may be nitric oxide production as well). Which buffer have you used for sorting? Please, remember, changes in the buffer, pH, ionic strength and, of course laser illumination during sorting all is stress factors. This is a possible reason.
Do I understand you correctly, Alexander, that BEFORE sort these macrophages do NOT make surplus NO? So you cannnot blame it on antibody injection? If so, than it has to have something to do with the physical insult on macrophages by the sort procedure or by way you tag your cells for sorting. To alleviate the former, try using the lowest stream velocities possible, and after sort let the cells recuperate for a while (even half-day under propoer conditions) before further testing. For the latter - depends what natibody you use to tag the macrophages - can it activate them?
Taras and Jacek, thank you for your posts. We checked and found that mAb staining does not activate macrophages. We have decided to do a simple experiment testing if putting macrophages through the sorter will result in their activation.
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