I imagine the response you can get from "older" monocytes depends on how you stored your sample and how viable/vital your cells are. If mantained in a in vitro culture all leukocytes will undergo apoptosis in relatively short times, and if mantained in the same medium for few day will eventually starve. All this could hamper the cytokine signal in a quantitative way and eventually shift your cytokine profile on the pro-inflammatory path. otherwise, your cytokine response mainly depends on the stimulus and activation state of your leukocytes so, giving a proper aspecific stimulus (e.g. PMI/ionomycin), your leukocytes may not change their cytokine response in significative ways. As usual, fresh leukocytes are better.
Interesting question. I agree up to a point with what has been said by Davide. I would like to add however, that the process of purifying the monocytes by itself is likely to cause cellular activation. The Ficolling of cells, centrifugation, washing, plating onto plastic, scraping off etc are all likely to stress cells and can result in release of chemokines and cytokines. Likewise the use of antibodies is well known to cause activation. This can be quite rapid. Some years ago I measured chemokine and cytokine mRNA responses in circulating monocytes and PBMNs drawn from patients at different time points during cardiac surgery and found that the process of surgical incision and pumping blood through a external heart bypass/oxygenator machine resulted in a v.rapid increase in chemokine and cytokines which returned to pre-op levels several hours later after completion of surgery - perhaps not that surprising . There is therefore some merit in allowing the cells to "rest" after trauma. The best thing to do, is to measure the chemokine / cytokine responses in the fresh and rested monocytes. What you may find, is that the basal levels of chemokines and cytokines are higher in fresh monocytes, and the effect on any subsequent stimuli is less marked. Against that, rested monocytes may have a lower basal level and a higher response, but as Davide mentioned they are more likely to be preapoptotic.
Ian Teo
- Badges
- Science method
- Similar topics
- Chemistry
- General Biochemistry
- Biochemical Methods