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Monocytes are cells that are still
able to differentiate into other cell types, like Kupfer cells.
Monocytes are very easy to stimulate. Even the smallest lipopolysaccharide (LPS)
contamination of the medium will activate them. And also the handling to isolate the
cells from blood can be enough to stimulate them. But it is also possible to stimulate the monocytes in whole blood. 1 µg/ml LPS
will activate monocytes to produce TNFa within 4 hours. Approximately 40% of the
cells will be positive for TNFa. Make sure that the blood doesn't contain EDTA.
This will prevent activation by holding all Ca2+ ions. Sodium heparin should do
fine.
When activating monocytes to detect the cytokines intracellularly
monensin is not used to block secretion of the protein. Monensin itself is already able to
stimulate the monocyte (Yamato, K. et al., 1989; Orlinska,
U. and Newton, R.C., 1992). This means that the specific signal does not have to
be higher than
the control (unstimulated sample). Or the cells start producing cytokines they wouldn't when they
were stimulated with the stimulus alone, but produce these because of the
secretion inhibitor.
Other ways to stimulate monocytes (except for LPS) are the ligation of CD40
by CD40L transfected cells or the soluble trimer of CD40L and of course the
combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin. The problem
of activated monocytes is they stick firmly to the culture well, especially when
using PMA/ionomycin. To get the
cells from the plate a rubber policeman/cell scraper can be used. Disadvantage is the fact that you
will loose a large number of cells. Maybe those that became vulnerable
during activation in the presence of the secretion inhibitor. The best way to
get the monocytes is treating them with 2 mM EDTA on a bed of ice. The EDTA
will bind the Ca2+, which the monocytes need to adhere. And the cold
will get them rounded, which make it easy to get them from the plate. A third solution is the use of
ultra low binding plates. These plates are coated in a way the monocytes can't
adhere in the starting hours of the culture. We tested them during several day
cultures. After three days the monocytes seem to eat the coating from the
plates. A third option is to use a commercial non-enzymatic cell dissociation
solution like Sigma sells. This works fine, also with keratinocytes, but
probably also contains EDTA.
IL-1ß The detection of interleukin-1 beta (IL-1ß) produced by monocytes asks for a
different approach than usual because the production of IL-1ß is post-transcriptionaly
regulated. The pro-IL-1ß is already present in the cytoplasm and clipped by ICE
to 'produce' the bioactive IL-1ß. There will always be a background
staining in the unstimulated sample and there is no point using a transport
inhibitor. The staining will be diffuse throughout the cell and is difficult to distinguish
from an aspecific binding of the antibody.
IL-18 The same results can be obtained by the search for the cytokine
IL-18. We regret the fact that there is only one company in the world that has
the right to sell anti-human IL-18. And they do it at a price that is
unrealistic. Many of us make the choice not to look into this cytokine.
IL-12 Also the detection of IL-12 is a special point of attention. We go into this
under dendritic cells.
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Monocytes
