I'm looking for a proper cell line and protocol to test the inhibition of a known plasminogen receptor. I'm thinking that exogenously adding plasminogen and then immunoblotting for cleaved plasmin would be an appropriate way to measure this. However, in this case the cells used would need to express tPA and/or uPA and I'm having a bit of trouble finding an appropriate cell line. Possibly a vascular endothelial cell line? Any suggestions would be helpful.
You can try a functional assay with HL-60 cells which express tpa avidly and thus adding plasminogen. It should generate plasmin that can be followed by chromogenic substrate S-2251, you can also do a Western of the supernatant and see the cleaved plasminogen. Fibroblast may work as well. I am attaching two papers one on platelets and one on fibroblasts. Follow the chromogenic substrate hydrolysis (450 nm yellow) as a function of time and stop the reaction (end point) with a solution of 50% acetic acid.
This is great information! Thank you very much for your answer.
There was actually only one paper attached to your answer. Would you mind pointing me in the direction of the second? I would greatly appreciate it. Thanks again!
Only one was uploaded by the system, here goes the second one, this one is on fibroblasts and looking for thrombin and FXa instead of plasmin. As you can see, the chromogenic substrate provides the specificity of the enzyme you are interested in measuring. You are lucky because there is one for plasmin S-2251.
You should separate the plasmin Generation Phase from the plasmin detection Phase. In the plasmin-detection Phase you should add about 1.5 mol/l Arginine (final conc.), pH 8.7 (final pH), 0.1 % Triton X100 (final conc.) to avoid Splitting of the chromogenic Substrate by kallikrein like enzymes.
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