OR
Hello Max,
I have a few suggestions to the protocol, feel free to incorporate them or point out any doubts that you have.
1. When choosing the microtiter plate you could also consider Maxsorp™ plates. Those are traditionaly used for adhesion assays with collagen and also for protein immobilization in quantitative ELISA.
2. When incubating the microtiter plate with PSGL-1, consider using 50 microliters of a highly concentrated solution of the protein (titrations of the protein would help to find the most suitable concentration for platelet adhesion after activation). Note that the incubation would benefit from an overnight sitting at 4ºC.
3. Further wash of the plates should be performed slowly afterwards, where PBS would be used as a washing buffer. Dumping could be performed, but a standard of good practice would require the usage of a multichannel pipette.
4. Platelet suspensions (equilibrated to the count of no less than 150 × 10^3 platelets / μl) should be pre-incubated with your P-selectin binding candidates (compounds) and further added to the test-wells.
5. Platelet activation should be carried simultaneously in each well, as you need to establish a standard timepoint for the assay. Platelet activation and P-selectin expression could be induced by thrombin (25nmol, perhaps). Please note that I am considering the platelet suspension as washed platelets in a neutral buffer, never use PRP. Also make sure to keep a standard incubation time and temperature for this stage.
6. Make sure you have control wells with Bovine serum albumin (BSA) or other non-PSGL-1 proteins, for non-specific binding. Adding a P-selectin inhibitor such as Anti-human CD62P should also be considered as a control for non-specific binding.
7. After the addition of thrombin, wash the wells again with the washing buffer (up to three times) and use a platelet-binding antibody such as FITC-antihuman CD31 to locate the platelets. Measuring fluorescence could give you either the microscopic picture of each well (Fluorescence microsocpy) or the possible quantitative inhibition of platelet adhesion reflected by an increase of fluorescence intensity in titrations of each test-molecule.*
*Note that you should add ~100μl of buffer to each well before scanning, to correct the optical path of the excitation lasers.
Alternatively, you could evaluate platelet binding through a colorimetric assay using a biotinylated anti-human CD31 antibody to mark platelets and a streptavidin-acetylcholinesterase system to be revealed after the addition of Ellman's reagent. Usually you could let the plate develop over the course of 1 hour and read it at 417nm.
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Please NOTE that for each experiment positive controls regarding max P-selectin binding and max fluorescence/colorimetric detection by the plate reader should be included.
GOOD LUCK! :)
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