BackgroundSince 1993, the Research and Development Group (R&D), at the Department of Clinical Chemistry, Ullevaal University Hospital, has had a routine for isolation (elutriation, centrifugation) and cryopreservation of human monocytes from whole blood for research purposes.
AimThis project’s aim has been to quality check a particular step (monocyte responsiveness to LPS stimulation) in this routine (see Background and Flow chart). Our personal aims have been the following: Get familiar with laboratory work, i.e. pipetting, ELISA quantification, flow cytometry and elutriation (sections 2.1, 2.2, 2.5). Learn how to evaluate data statistically (2.3). Acquire knowledge of the monocyte’s role in atherogenesis and sepsis (2.4).
Materials and methodsIsolated and cryopreserved monocytes were thawed and stimulated with LPS. In parallel non-stimulated monocytes were examined. The supernatants were subsequently analysed for TNF-alpha by use of a commercial ELISA kit.In a separate experiment we measured TNF-alpha in heparinplasma from in vitro LPS-stimulated whole blood.
ResultsWe showed that LPS-stimulated monocytes produced TNF-alpha(mean: 1200 pg/ml/12.500 cells), while unstimulated monocytes did not. TNF-alpha production was higher in in vitro LPS stimulated whole blood (mean: 8000pg/ml) than in supernatants from LPS stimulated isolated monocytes. The TNF-alpha production of monocytes from different donors differed (Range: 1000-1800 pg/ml/12.500 cells) although they were treated in a similar manner. Our results became better as we practised and built up our skills (Variation Coeff. down). Our TNF-alpha measurements were comparable, but our results tended to lie systematically lower than the results from the R&D-group.
DiscussionThe fact that monocytes produced TNF-alpha as a response to LPS stimulation, while unstimulated did not, indicated that the isolation and cryopreservation routine had been successful, and that the monocytes kept their viability. Probable explanation for the higher TNF-alpha production in whole blood: more monocytes were stimulated by a higher LPS concentration.The donor variation could be explained by analytical or biological variations. The R&D-group also found donor variations. They have grouped their samples into “high-” and “low-responders”. Since no longitudinal cell controls were run, no final conclusion as to donorvariation could be given.The reason why our results systematically lie under the R&D-groups` is probably because we used supernatants that were frozen stored and thawed, while the R&D-group used fresh supernatant (T1/2 TNF-alpha).