We are trying to develop an assay demonstrating the death primary human cells as a result of the membrane attack complex. Our source of complement proteins is normal human serum. We are in need of a positive control which induces cytolysis through the alternative complement pathway. Any advice will be appreciated.
Dear Carson,
There are several ways to do that (at least in theory).
Working with Mg/EGTA serum you can:
1. Block surface regulators with antibodies (CD46, CD55, CD59) or cut them off using enzymes that cleave GPI-anchor
2. Use neuraminidase to remove sialic acids that attract factor H to cell surfaces.
3. Use heme/hemin as described here: Article Complement activation by heme as a secondary hit for atypica...
4. Pre-opsonize your cells with C3b. This one is very artificial.
We are working a bit with heme and it seem to work but cells don't like it of course, appropriate concentrations should be used.
Hope that can help.
All the best and good luck!
Dear Carson,
Zymosan is a very potent activator of the alternative complement pathway. In our hands zymosan + human serum efficiently enhances generation of complement activation products (anaphylatoxins) and TCC deposition on primary cells.
However, we observed that isolated cells strongly upregulate protective surface markers such as CD59.
Therefore we see cell death mainly in tissue culture (not only lysis, more regulated cell death we guess) but hardly in isolated cells.
Overall, cellular response of isolated cells is quite different from that of native material.
Depending on your project, you might consider characterization of cell surface markers associated with complement regulation/ protection. Maybe your cells are "resistent".
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