Hanging Drop Aggregation Assay - How to interpret? (Sep/11/2009 )
I am interested in doing an haning drop aggregation assay. I was wondering what is the principle behind this assay.. I thought that when u have cells in a drop and hang it upside down, obviously all cells will aggregate as a result of gravity. How does this assay tell us the aggregation property of cancer cells.
I was wondering if this assay does tell us that, can I use an antibody to my protein of interest to see if there is no aggregation? Or have a dose response to my protein to see to see the rate of aggregation? Thanks all!!!!
I'm also interested in using this assay. I was reading some papers in which this method was employed but I don't understand one part of the procedure. Maybe this is because I'm missing something behind the basis of the method.
My doubt is: for quantification, some protocols say "Resulting cell clusters were subjected to 30 rounds of pipetting through a 200-ul Gilson pipette, and the degree of dissociation was quantified by counting the particles after trituration". I don't get this part...
So, after the incubation the drops/clusters of cells are still in the lid of the plate, right? What am I supposed to do with them?
And another question: how many drops per well should I put in the lid of a 24-well plate?