I want to do a monocyte binding assay under static conditions in a matrigel matrix. My problem is to get rid of unbound monocytes after an adequate incubation time so I just have bound monocytes on endothelial cells left in the gel. I tried different matrigel concentration, but the lower the concentration the more problems I have with the washing steps (losing gel as well as all cells). Is the matrigel too thick, my monocytes don't bind completely to the endothelial cells. The bound monocytes have to be measured with flow cytometry (the protocol for that is already established - the matrigel is the problem). Has anyone ever tried a similar experiment or is really experienced in using matrigel or other ECM? Thanks for helping me.
Hello Alexander,
yes, it has to be 3D. The endothelial cells are embedded in the matrigel and the monocytes (labelled for flow cytometry later) will be pipetted on top of the endothel cell - matrigel - layer and incubated for 2 h before i have to wash all unbound monocytes away, which works perfectly in a 2D experiment but not with the matrigel. The concentration of matrigel is very low (1mg/ml) what makes it difficult to wash.
The gel isn´t solid at all at this concentration. The problem is that i can´t really say how much of the monocytes already migrated into the gel and bound properly to the endothelial cells and how much monocytes maybe migrated into the gel but didn´t bound (like stuck on their way to the ECs but didnt reached them in time) ... the fluorescent microscope is useless because of the thickness of the gel layer. You see, its a lot of trouble with a 3D version of this experiment. Maybe i can use heparin but i have to discuss this with my supervisor to make sure that heparin is not interferring with the whole concept (TNFa, irradiation...).
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