Has anybody done Complement Mediated Cytotoxicity?

My name is Andrew Zhang and I am a new poster on this forum.

I am trying to do a complement mediated cytotoxicity experiment using the human cell line LNCAP (prostate cancer cells) and using anti-PSMA (prostate specific membrane antigen) antibodies. LNCAP cells express high levels of PSMA.

In my assay, I am using goat anti-human PSMA polyclonal IgG at a concentration of 10 ug/ml, and then following up with 20% goat complement preserved serum in HEPES buffer (25 mM HEPES, 150 mM NaCl, 1.5% BSA, 5 mM glucose, and 1.5 mM MgCl2). In addition, I am using mouse anti-CD46 and anti-CD59 antibodies as blockers for complement inhibitors. As a positive control I am using 2% cetyltrimethylammonium bromide in the 20% serum solution for max cell killing. As a negative control I am just incubating the cells with serum without any antibodies. I am trying to look at cell killing via flow cytometry with propidium iodide. Basically, what I did was after I incubated my cells (2x10^5) with the serum and antibodies, I added propidium iodide to a final concentration of 1 ug/ml and analyzed using flow cytometry (FACSCalibur). Strangely, I get positive signal (~10^3) for the negative control and the samples containing antibodies (same fluorescence level) but no signal at all for the cetyltrimethylammonium bromide experiment.

Can anybody with experience in this area provide some insight? Thanks so much! -Andrew

EDIT: PS. I am trying to do antibody dependent cell-mediated cytotoxicity too. It is also against LNCAP with anti-PSMA antibodies, but this time I am using a mouse IgG2b with J774A.1 mouse macrophage. Has anybody had any experience with ADCC? Thanks again!

Edited October 4, 200817 yr by azhang
forgot to write something