Materials and Reagents

1. Pipette tips 0.1-10 μl (Eppendorf, epT.I.P.S.^® Reloads, catalog number: 022491504 )
2. Pipette tips 0.5-20 μl (Eppendorf, epT.I.P.S.^® Reloads, catalog number: 022491521 )
3. Pipette tips 2-200 μl (Eppendorf, epT.I.P.S.^® Reloads, catalog number: 022491539 )
   
4. Pipette tips 50-1,000 μl (Eppendorf, epT.I.P.S.^® Reloads, catalog number: 022491555 )
5. 15 ml Falcon tube (Corning, catalog number: 352096 )
6. Tygon 3350 silicone tube (Saint-Gobain Performance Plastics, catalog number: ABW00002 )
7. Safety-Multifly-Needle 21G (SARSTEDT, catalog number: 85.1638.935 )
8. Membrane adaptor (SARSTEDT, catalog number: 14.1112 )
9. 5 ml syringes (BD, Discardit II^TM, catalog number: 301285 )
10. 50 ml syringes (BD, perfusion, catalog number: 300136 )
11. Bottomless 6 channel sticky slide (IBIDI, sticky slides VI^0.4, catalog number: 80608 )
12. Glass coverslips 24 mm x 24 mm (SCHOTT, NEXTERION^®, No. 1.5, catalog number: D263T )
13. Cuvette (Eppendorf, catalog number: 952010069 )
14. Human blood (taken from male and female healthy donors with the age of 25-40 years old)
15. Plasmid ptdTomato (Takara Bio, catalog number: 632531 )
16. E. coli strains (DH12S, Invitrogen)
    E. coli was transformed with plasmid ptdTomato. TdTomato is encoded within the lac operon and transcription is induced in the presence of isopropylthio-β-galactoside (IPTG) (IPTG binds to the lac repressor and releases lac repressor from the lac operator). The plasmid carries the bla-gene for ampicillin resistance.
    Note: E. coli stock was made in LB with 7% dimethyl sulfoxide (v/v) and kept at -80 °C.
17. Distilled water (ddH₂O) (Millipore)
18. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2438 )
19. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D8537 )
20. Recombinant human albumin (rHSA) (Sigma-Aldrich, catalog number: A9731 )
    Note: Dissolve rHSA in ddH₂O to 8% (w/v) solution and store the aliquots at -20 °C.
21. Prostaglandin I₂ sodium salt (PGI₂) (Abcam, catalog number: ab120912 )
    Note: Dissolve it in DMSO to 10 mg/ml and store the aliquots at -20 °C.
22. HEPES solution (Sigma-Aldrich, catalog number: H0887 )
23. 20% HNO₃ (Carl Roth, catalog number: 4337.2 )
24. Hexamethyldisilazane (HMDS) (Sigma-Aldrich, catalog number: 440191 )
25. Fibrinogen from human plasma (Sigma-Aldrich, catalog number: F3879 )
    Note: Dissolve it in 0.9% NaCl to 2 mg/ml and store the aliquots at -20 °C.
26. Fibrinogen from human plasma, Alexa Fluor 488 conjugate (Thermo Fisher Scientific, Life technology, catalog number:    F13191 )
    Note: Dissolve it in 0.1 M NaHCO₃ (pH 8.3) to 1.5 mg/ml and store the aliquots at -20 °C.
27. U46619 (Enzo Life Sciences, catalog number: BML-PG023-0001 )
    Note: Dissolve it in DMSO to 28.8 mM and store the aliquots at -20 °C.
28. Adenosine 5′-diphosphate sodium salt (ADP) (Sigma-Aldrich, catalog number: A2754 )
    Note: Dissolve it in PBS to 20 mM and store the aliquots at -20 °C.
29. Thrombin from bovine plasma (Sigma-Aldrich, catalog number: T4648 )
    Note: Dissolve it in 0.1% (w/v) bovine serum albumin (BSA) to 100 U/ml and store the aliquots at -20 °C.
30. LB broth (Sigma-Aldrich, catalog number: L3022 )
    Note: 20 g LB powder was dissolved in 1 L ddH₂O and autoclaved.
31. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, catalog number: I6758 )
    Note: Dissolve IPTG in ddH₂O to 100 mM and store the aliquots at -20 °C.
32. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518 )
    Note: Dissolve ampicillin in ddH₂O to 50 mg/ml and store the aliquots at -20 °C.
33. Sodium citrate tribasic dehydrate (C₆H₅Na₃O₇•2H₂O) (Sigma-Aldrich, catalog number: S4641 )
34. Citric acid monohydrate(C₆H₈O₇•H₂O) (Sigma-Aldrich, catalog number: C1909 )
    
35. Glucose (Merck, Calbiochem, catalog number: 346351 )
36. Sodium chloride (NaCl) (Carl Roth, catalog number: 3957.3 )
    
37. Sodium bicarbonate (NaHCO₃) (Merck, catalog number: 106329 )
    
38. Potassium chloride (KCl) (Carl Roth, catalog number: 6781.3 )
39. Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C8106 )
    Note: CaCl₂•2H₂O was dissolved in ddH₂O to make 100 mM stock solution and stored at 4 °C.
40. Acid-citrate-dextrose (ACD) buffer, pH 4.7 (see Recipes)
41. Modified Tyrode's buffer (see Recipes)
42. Fibrin buffer (see Recipes)
    

Equipment

1. Pipette 0.1-2.5 μl (e.g., Eppendorf, Research plus: O18485E)
2. Pipette 0.5-10 μl (e.g., Eppendorf, Research plus: O30964E)
   
3. Pipette 10-100 μl (e.g., Eppendorf, Research plus: O30379E)
4. Pipette 100-1,000 μl (e.g., Eppendorf, Research plus: O29260E)
5. Orbi-Shaker (e.g., Benchmark)
6. pH meter (e.g., Mettler Toledo)
7. BioPhotometer (e.g., Eppendorf, model: BioPhotometer 6131 )
8. Bunsen burner (e.g., Campingaz)
9. Centrifuge (e.g., Eppendorf, model: 5804 )
10. Cell culture incubator (e.g., Binder)
11. Hematology counter (e.g., Horiba Medical)
12. KLM spin coater (e.g., Schaefer)
13. Inverted phase contrast and fluorescent microscope (e.g., OLYMPUS, model: IX83 )
14. Syringe pump (e.g., KD Scientific, model: KDS 100 )
    

Software

1. Live cell imaging software that was connected to the microscope (e.g., CellSense)
2. Fiji software [National Institutes of Health (NIH)]
   

Procedure