UV-Visible Spectroscopy for Monitoring Fibrin Clot Formation

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1. Preparation of Aβ42 and Fibrinogen for Analysis

1. Prepare monomeric Aβ42 from lyophilized powder
   1. Warm Aβ42 powder to room temperature and spin down at 1,500 x g for 30 s.
   2. Add 100 µL of ice-cold hexafluoroisopropanol (HFIP) per 0.5 mg of Aβ42 powder and incubate for 30 min on ice.
      CAUTION: Use care when handling HFIP and perform all steps in a chemical hood.
   3. Prepare 20 µL aliquots and let the films air-dry in a chemical hood for 2-3 h. Films can be stored at -20 °C.
   4. Reconstitute monomeric Aβ42 film in 10 µL of fresh dimethyl sulfoxide (DMSO) by agitation in a bath sonicator for 10 min at room temperature.
   5. Add 190 µL of 50 mM tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.4) and pipette up and down gently.
   6. Remove the protein aggregates by centrifugation at 20,817 x g at 4 °C for 20 min.
   7. Incubate the supernatant at 4 °C overnight and the next day centrifuge the solution at 20,817 x g at 4 °C for 20 min to discard any further protein aggregates.
   8. Measure Aβ42 concentration by bicinchoninic acid (BCA) assay. Serial dilute purified bovine serum albumin (BSA) from 1 mg/mL to 0.0625 mg/mL to produce a protein standard, and add 10 µL of each standard to the wells of a multi-well plate in triplicate. Dilute Aβ samples 1:4 and add 10 µL to the wells in triplicate. Mix BCA solutions A and B and add 200 µL to each well. Incubate for 30 min at 37 °C and read on a plate reader at 562 nm. Keep the remaining Aβ solution on ice and use this preparation for turbidity assay and SEM.
2. Prepare Fibrinogen solution
   1. Measure 20 mg of lyophilized fibrinogen powder into a 15 mL tube and re-suspend with pre-warmed 2 mL of 20 mM hydroxyethylpiperazine ethane sulfonic acid (HEPES) buffer (pH 7.4).
   2. Incubate in a 37 °C water bath for 10 min.
   3. Filter the solution through a 0.2 µm syringe filter and store at 4 °C for 30 min. Take out the solution from 4 °C and filter again through a 0.1 µm syringe filter to remove fibrinogen aggregates or pre-existing fibrin.
   4. Measure the fibrinogen concentration by BCA assay. Follow the instructions from Step 1.1.8. Keep the remaining fibrinogen solution at 4 °C and use this preparation for turbidity assay and SEM.

2. Clot Turbidity Assay

1. To each experimental well of the 96-well plate, add 20 µL of 30 µM Aβ42 solution from Step 1.1.8 so that its final concentration is 3.0 µM in 200 µL of the buffer. Add the same volume of 5% DMSO in 50 mM Tris buffer (pH 7.4) to buffer control wells in the 96-well plate.
   NOTE: The exact volume of Aβ42 at this step is not important. For low-concentration preparations, a higher volume can be used, and the volume of the clot formation buffer can be adjusted. The final volume should remain at 200 µL.
2. If testing inhibitory compounds, dilute the compound to the working concentration as DMSO. Add compound or DMSO alone for a control to the wells with Aβ42 and mix well.        
   NOTE: Aβ42 solution should form a discrete droplet at the bottom of the well, the compound or DMSO should be pipetted directly into the center of the droplet.
3. Dilute the fibrinogen stock solution from Step 1.2.4 in clot formation buffer (20 mM HEPES buffer (pH 7.4), 5 mM CaCl₂, and 137 mM NaCl) and add it to Aβ42 containing and control wells of the 96-well plate. Adjust the volume of the fibrinogen solution so that its final concentration becomes 1.5 µM in total 200 µL reaction volume. Pipette the solution slowly and avoid forming bubbles. Incubate the plate at room temperature for 30 min shaking on a rotating platform.
   NOTE: The volume of fibrinogen solution may vary depending on its stock concentration, but the total volume in each well should be 170 µL while incubation.
4. Prepare thrombin solution by dissolving commercially purified thrombin powder in ddH₂O to make a stock solution of 50 U/mL. Dilute to the 5 U/mL working solution in 20 mM HEPES buffer (pH 7.4) directly before use.
5. After 30 min of incubation, simultaneously add 30 µL of thrombin solution (5 U/mL) into the fibrinogen solutions in the 96-well plate from Step 2.3 using a multichannel pipette. The addition of thrombin will immediately initiate clot formation. Therefore, add thrombin directly into the center of the fibrinogen solution with care to avoid forming bubbles.    
   NOTE: The activity of thrombin can vary between experimental conditions, as well as thrombin lots. The correct concentration required to robustly produce clots may have to be determined empirically.
6. Read the absorbance of in vitro clot on a plate reader immediately following the thrombin addition. Measure the absorbance at 350 nm over the course of 10 min, every 30-60 s. Perform the entire assay at room temperature.
   NOTE: Some inhibitor compounds may alter the solution absorbance at 350 nm, in which case the turbidity can be measured at 405 nm.

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자료

Name	Company	Catalog Number	Comments
Fibriogen, Plasminogen-Depleted, Plasma	EMD Millipore	341578	Keep lid parafilm wrapped to avoid exposure to moisture
Beta-Amyloid (1-42), Human	Anaspec	AS-20276
Thrombin from human plasma	Sigma-Aldrich	T7009
1,1,1,3,3,3-Hexafluoro-2-propanol, Greater Than or Equal to 99%	Sigma-Aldrich	105228
Dimethyl sulfoxide (DMSO), sterile-filtered	Sigma-Aldrich	D2438
Pierce BCA Protein Assay Kit	Thermo Scientific	23225
Tris Base	Fischer Scientific	BP152
HEPES	Fischer Scientific	BP310
NaCl	Fischer Scientific	S271
CaCl2	Fischer Scientific	C70
Filter Syringe, 0.2µM, 25mm	Pall	4612
Millex Sterile Syringe Filters, 0.1 um, PVDF, 33 mm dia.	Millipore	SLVV033RS
Solid 96-Well Plates High Binding Certified Flat Bottom	Fischer Scientific	21377203
Spectramax Plus384	Molecular Devices	89212-396
Centrifuge, 5417R	Eppendorf	5417R
Lab Rotator	Thermo Scientific	2314-1CEQ