Procedures A. Flow chamber preparation and whole blood perfusion
A1. Preparation of coverslips
CRITICAL. Wash cleaned coverslips thoroughly with water.
1. Degrease new coverslips (use tweezers) with 2 M HCl in 50% ethanol.
2. Wash coverslips twice with milliQ water to remove residual HCl.
3. Leave coverslips to dry on drying rack.
A2. Coating of coverslips
CRITICAL. Prevent drying of biological material on coverslip by storing in humid environment.
4. Prepare coating material (collagen, collagen peptide, decorin, fibrinogen, fibronectin, laminin, osteopontin, rhodocytin, thrombospondin, vitronectin, vWF), at 50-250 μg/mL, as described.9
5. Mount coverslip onto precision mall for coating.
6. Apply 0.5 µL of coating solution(s) in the assigned place(s), and remove from the mall.
7. Store coverslip in humid environment to prevent drying out of microspots. Allow coating material to bind for 60 minutes at room temperature.
8. Block uncoated glass with blocking buffer, and leave in humid environment for 30 minutes.
9. Wash blocked coverslip with saline. If not immediately used, leave coverslip in humid environment to prevent drying.
A3. Assembly of coverslip and flow chamber
CRITICAL. Check for leakage of the mounted flow chamber. Also check rigorously for absence of air bubbles before starting the experiment. Note that temperature changes can lead to appearance of air bubbles. Keep inlet tubing as short as possible
10. Connect tubing to inlet and outlet of the flow chamber.
11. Rinse chamber and tubing with flow buffer, check for absence of air bubbles, and mount coated coverslip on top of chamber.
12. Place chamber with coverslip in aluminium holder and tighten screws.
13. Check that flow chamber system is leak-tight by perfusion with flow buffer, flush out any bubbles in chamber.
A4. Drawing of human blood by venipuncture
CRITICAL: Before drawing human blood, obtain permission from your Medical Ethical Committee, according to the local and national regulations, and get full informed consent from donor. Coagulation (traces of thrombin) need to be rigorously prevented by drawing without constraints, mixing well with anticoagulant, and incubation at 37oC. Note that PPACK is only shortly active as an anticoagulant at neutral pH. Other possible errors are described elsewhere.4
14. Add 0.5 mL saline into a 5 mL polystyrene blood collection tube. Add 40 U/mL (f.c.) fragmin, and add 40 µM PPACK (f.c.) just before venipuncture.
15. Draw blood according to local protocols, and let smoothly flow into collection tube. Directly mix blood with anticoagulant solution. We prefer an open system using a 23 gauge needle, to ensure undisturbed flow. Discard first 1 mL of blood before filling blood collection tube.
16. Incubate the collected blood at 37°C for 10-15 minutes to allow platelets to resensitize.
17. Preferably, determine platelet and red cell counts. A decrease in platelet count points to aggregation of platelets, e.g. by traces of thrombin.
18. Add additional 20 µM PPACK (f.c.) once per hour.
A5. Blood perfusion through flow chamber
CRITICAL: Check for correct pump settings to obtain the requested shear rate. Check for absence of air bubbles and fibrin clots during the experiment. Preferably use an inverted microscope. Several possible errors are described elsewhere.4
19. Only for experiments to determine stable platelet adhesion or thrombus volume, add 0.5 µg/mL DiOC6 (f.c.) to 0.5 mL blood sample. Allow staining of the blood cells for 5 minutes.
20. Draw 0.5 mL blood sample into 1 mL syringe equipped with blunt needle. Make sure that no air is present in top of needle.
21. Connect syringe with blood sample to inlet tubing of prepared flow chamber. Carefully avoid air bubbles (fluid-fluid contact).
22. Prepare 1 mL syringe with flow buffer with required fluorescent labels. The following labels are suitable for post-staining: FITC-labeled anti-CD62P mAb (1.25 µg/mL) or FITC-labeled anti-fibrinogen mAb (1:100) and/or AF647-labeled annexin A5 (0.25 µg/mL) (all f.c.).
23. Mount flow chamber and holder on stage of the microscope, and identify position of microspots with camera. Focus on optical plane of one microspot.
24. Place syringe filled with whole blood on perfusion pump (push mode). Ensure proper pump settings (arterial wall shear rate: 1600 s-1 for 3.5 minutes or 1000 s-1 for 4 minutes; venous wall shear rate 150 s-1 for 6 minutes). Note that the wall shear rate depends on the flow rate and the dimensions of the flow chamber. For calculation, see elsewhere.4
25. Switch pump on. The experiment starts when the blood has reached the site of the microspots.
A6. Recording of stable adhesion and thrombus volume using DiOC6-labeled platelets
CRITICAL: Rinse shortly to prevent disaggregation of thrombi.
26. During the first 2 minutes of whole blood perfusion, record DiOC6 fluorescence microscopic images at 2-seconds intervals (real-time recording of stable platelet adhesion).
27. After 3.5, 4 or 6 minutes of perfusion (depending on shear rate), change syringe with blood by syringe with flow buffer; set pump rate at 1000 s-1.
28. For 2 minutes, rinse chamber with flow buffer.
29. Take confocal z-stacks from thrombi during stasis; 2 to 3 stacks per microspot (recording of thrombus volume).
A7. Recording of brightfield images and post-staining with fluorescent labels
CRITICAL: Rinse shortly to prevent disaggregation of thrombi. Prevent air bubbles in flow chamber during syringe replacements.
30. Starting from point 25.
31. At the end of the whole-blood perfusion, change syringe with blood by syringe with fluorescent labels; set pump rate at 1000 s-1.
32. For 2 minutes, perfuse buffer with labels through flow chamber; leave 1 minute for staining.
33. During the perfusion, take brightfield microscopic images (5 per microspot) under flow.
34. Change syringe with labels by syringe with flow buffer; and perfuse for 2 minutes to remove unbound label.
35. Take fluorescence images during stasis (5 images per microspot).
Procedures B. Brightfield and fluorescence microscopic imaging of thrombi
B1. Use of LSM 7 LIVE line-scanning confocal fluorescence microscope
SPECIFICATION: Recording of stable platelet adhesion and thrombus volume (DiOC6-labeled platelets). Use in confocal mode for rapid real-time scanning of platelet adhesion, and of z-stacks to determine thrombus volume (see also Ref.12). Collect only sharp, high-quality images! Three color staining is possible (excitations 485, 530, 640 nm).
1. Microscope: inverted confocal fluorescence microscope: Axio Observer Z1 (Carl Zeiss) with differential interference contrast (DIC) optics. Camera: AxioCam HRm (Zeiss). Scanning stage with insert for flow chamber holder.
2. Laser head: LSM 7 Live (Zeiss). Lasers: DOPP 488 nm (100 mW), DPSS 532 nm (75 mW), Laser 635 nm (30 mW).
3. Objective: 63x oil immersion (Zeiss, PlanApo, NA 1.40; DIC M27, WD 0.19 mm).
4. Settings: [configuration 488 laser line]
a. Excitation 488 nm, emission filter 495-555 nm, pinhole 1 AU.
b. For time series: 1 cycle of 2 minutes with 2-seconds interval, laser power 5%, gain 5, zoom 0.5x, scan speed 3-4 Lps.
c. For z-stack: 0.5 µm between optical planes (70 slices), laser power 5%, gain 5, zoom 1x, scan speed 1 Lps.
5. Controlling software: ZEN 2010 (Zeiss).
6. Output images: LSM file (512 x 512 pixels, 107 x 107 µm or 213 x 213 µm (depending on zoom), 8-bit).
B2. Use of BioRad/Zeiss Radiance 2100 laser scanning confocal microscope
SPECIFICATION: Imaging of thrombi post-labeled with FITC (OG488) and AF647 probes. Flow chamber with labeled thrombi is placed on stage up-side down. Scan with large pin holes to collect fluorescence from all optical planes. Collect only sharp, high-quality images! Three color staining is possible (excitations 485, 530, 640 nm).
7. Microscope: right-up fluorescence microscope E600FN (Nikon, Japan). Scanning stage with insert for flow chamber holder.
8. Laser head: BioRad/Zeiss scan head. Lasers: Argon 488 nm (40 mW), Green He/Ne 543 nm (1.5 mW), Red diode 638 nm (5 mW).
9. Objective: 60x oil immersion (Nikon, PlanApo SFluor, NA 1.30, WD 0.22 mm).
10. Settings: Two-color fluorescence:
a. PMT1: excitation 488 nm, laser power 20%, iris 1.5, emission filter 508-523 nm
b. PMT2: excitation 637 nm, laser power 50%, iris 3.5, emission filter >660 nm
c. zoom 1, Kalman averaging 2, scan speed 160 Lps.
11. Recording software: LaserSharp 2000 software (Zeiss).
12. Output images: PIC file (512 x 512 pixels, 200 x 200 µm, 8-bit).
B3. Use of camera-based non-confocal fluorescence microscope system
SPECIFICATION: Imaging of thrombi post-labeled with FITC (OG488) and AF647 probes. Furthermore, recording of brightfield phase-contrast images to determine platelet deposition. Collect only sharp, high-quality images!
13. Microscope: inverted fluorescence microscope Diaphot 200 (Nikon) with phase-contrast. Two cameras connected with beam splitter, post-magnification and removable infrared filter. Vista brightfield CCD camera; Hamamatsu EM-CCD C9100-12 fluorescence camera. Scanning stage with insert for flow chamber holder.
14. Fluorescence: Xenon lamp (100 W). Filter cube: FITC (OG488): exciter 485 ± 11 nm, dichroic 400 nm, emitter 530 ± 15 nm. Brightfield trans-illumination (white light).
15. Objective: 40x oil-immersion (Nikon, Fluor/100, NA 1.30. Ph4DL, WD 0.20 mm).
a. Brightfield phase-contrast (empty filter cube). Post-magnification: 1x
b. Fluorescence: excitation 485 nm, emission 530 nm. Post-magnification: 1.5x.
17. Recording software: Axiovision 4.8 (Zeiss).
18. Output images: TIFF file (512 x 512 pixels, 200 x 200 µm, 8-12 bit).
B4. Use of EVOS table fluorescence microscope
SPECIFICATION: Imaging of thrombi post-labeled with FITC (OG488) and AF647 probes. Furthermore, recording of brightfield images to determine platelet deposition (overlays can be made). Collect only sharp, high-quality images! Three color staining is possible (excitations 485, 530, 640 nm).
19. Microscope: EVOS-FL, inverted microscope, infinity-corrected fluorescence optical system.
20. LED diodes: DAPI 357 nm (emission 447 nm), GFP 470 nm (emission 510 nm), RFP 531 nm (emission 593 nm), Cy5 626 nm (emission 692 nm). Brightfield trans-illumination (white light).
21. Objective: 60x oil immersion (Olympus, UPlanSApo, NA 1.35, WD 0.15 mm).
22. Settings: adjustable intensity of LEDs
a. Brightfield: transmitted light at intensity of 50%.
b. GFP cube: excitation 470 nm, emission 510 nm, intensity 40%.
c. Cy5 cube: excitation 626 nm, emission 692 nm, intensity 20%.
23. Recording software: integrated in EVOS system. Make sure to save images of individual colors.
24. Output images: TIFF file (1360 x 1024 pixels, 142 x 107 µm, 8-bit).
Procedures C. Analysis of brightfield and fluorescence images
C1. Image analysis for morphological score
CRITICAL: Analysis of images blinded for the experimental condition.
1. Determine morphological score of thrombi on coverslip based on recorded brightfield phase-contrast or DIC images.
2. Score at a 5-point scale (see Fig. 1).
C2. Image analysis with package Metamorph (Molecular Devices)
CRITICAL: Measurement of surface area coverage of brightfield and fluorescence images. The following procedures apply to 8-bit TIFF and PIC images. Conversion to 8-bit images can be done using ImageJ software (Open access). Image analysis can also be performed with ImageJ. Output data are given as numbers of pixels per region. To determine surface area coverage, use total pixel number of images. Note that the outcome of the analyses depends on the quality of the recorded images.
1. Protocol for stable platelet adhesion (see Fig. 2)
a. Threshold every image within one time series with the same threshold → binary image.
b. In “process” and “arithmic”, choose the first binary image as “source image 1” and the second binary image as “source image 2”.
c. Click “subtract” with constant values at “0”.
d. Choose apply.
e. Repeat steps a-d for the next images.
f. Choose “measure” → “integrated morphometry analysis”. Measure all binary images and subtracted images.
g. Export all values to Excel spreadsheet, and calculate % of change between consecutive images.
2. Protocol for surface area coverage of aggregated platelets (see Fig. 3A)
a. For each image, use edge detection in both horizontal (150) and vertical (150) direction.
b. Use the horizontally filtered image for threshold setting → binary image.
c. Use morphological close filter (diamond, width = 12) and open filter (circle, diameter = 5).
d. Transfer regions to brightfield/fluorescence image, and check if region detection is right.
e. Export data to Excel file, and convert pixel numbers to % surface-area-coverage.
3. Protocol for surface area coverage of platelet monolayers (see Fig. 3B)
a. Filter images using morphological bottom hat filter (diamond, width = 15), then close filter (diamond, width = 4).
b. Threshold closed image binary image.
c. Apply morphological dilate filter (square, width = 2).
d. Transfer regions to original image, and check if region detection is right.
e. Export data to Excel file, and convert pixel numbers to % surface-area-coverage.
4. Protocol for integrated feature size
a. The integrated feature size (IFS) is a value taking into account the proportional contribution of large and small thrombi on microspots. It represents the cumulative contribution of squared features, ranked from small to large individual features, with (f) from small to large are numbered 1-N. For formula see Ref. 9.
b. First, from an analyzed image, rank the individual features (pixels per region) from small to large (one image = one column with features) in an Excel file.
c. Determine the pixel size of one single platelet. Exclude all features smaller than this size (≈ 100 pixels).
d. Integrate the values of the features (accumulated sum of pixels).
e. Convert pixel size into µm2.
f. Divide the accumulated feature size by the accumulated sum of all feature sizes, and express as percentage.
g. Calculate the area above the percentage curve in µm2.
h. Express results on a logarithmic scale.
C3. Image analysis with package Axiovision 4.8 (Zeiss) for thrombus volume
CRITICAL: This program uses LSM files, and allows writing of scripts for automated image analysis. Common output is: ID region, volume unscaled (pixel3), surface (µm2) and volume (µm3) per region. Summative data can be calculated per region.
1. Use scrap filter with minArea: 1 and maxArea: 100 (see Fig. 3C).
2. Use separation filter with count: 3 and in Morphology mode.
3. Transfer regions to original image, and check if region detection is right.
4. Export data to Excel file, and convert pixel numbers to µm3.