Matrix degradation assay - (Aug/28/2008 )
I read a short M&M section about this method but do not understand what to measure;
they used Oregon Green 488 gelatin and fibronectin in PBS 2 M urea (what is urea for??); they quenched with borohydride (why?); they allowed cells for 24h to degrade this matrix; I think the Oregon Green will be measured but what is to expect? non-fluorescent holes in an microscopic image which represent degragation of gelatin/fibronectin?
please help to explain
-The Bearer-
QUOTE (The Bearer @ Aug 28 2008, 05:34 PM)
I read a short M&M section about this method but do not understand what to measure;
they used Oregon Green 488 gelatin and fibronectin in PBS 2 M urea (what is urea for??); they quenched with borohydride (why?); they allowed cells for 24h to degrade this matrix; I think the Oregon Green will be measured but what is to expect? non-fluorescent holes in an microscopic image which represent degragation of gelatin/fibronectin?
please help to explain
they used Oregon Green 488 gelatin and fibronectin in PBS 2 M urea (what is urea for??); they quenched with borohydride (why?); they allowed cells for 24h to degrade this matrix; I think the Oregon Green will be measured but what is to expect? non-fluorescent holes in an microscopic image which represent degragation of gelatin/fibronectin?
please help to explain
Good questions! I really don't know why they used urea or why they quenched. Did they want to measure some kind of phagocytosis of the gelatin, because then they would be able to see internalized Oregon green 488 in lyzosomes (especially if they quenched on the outside)? Fibronectin is composed of two similar polypeptide chains which are attached by disulfide bridges, which are broken by the urea. Fibronectin could inhibit binding to the gelatin by cells since they bind on the same receptors (probably), but I don't understand why they haven't just omit it then. Could you tell something more about the aim of this study?
-aspergillie-