Fibrinolysis_Data_Bead_Conc.xlsx

Data are results from experiments investigating fluoresnce labeling on fibrinolysis. Samples and analysis were prepared and performed in the following way: <br> On a glass microscope slide a small amount of optical glue (Norland Optical Adhesive 81, Norland Products, Cranbury, NJ), pressed under a PDMS stamp, was cured under ultraviolet light, resulting in a ridged surface with gaps between ridges measuring 20 μm wide and 10 μm deep. Aliquots of peak 1 fibrinogen (Enzyme Research Laboratories, South Bend, IN), ALEXA-488 fibrinogen (Invitrogen by Thermo Fisher Scientific/Life Technologies Corporation, Eugene, OR), and human alpha thrombin (Enzyme Research Laboratories, South Bend, IN) were thawed from -80°C. In a microcentrifuge tube, fibrinogen was diluted to 0.6 mg/mL with a HEPES Buffered Saline (HBS; 20 mM HEPES, 150 mM NaCl, pH 7.4) and mixed with ALEXA-488 fibrinogen at 1/65th the fibrinogen concentration. Separately, the thrombin aliquot was diluted to 2 U/mL using HBS with CaCl2. 10 µL of fibrinogen solution was pipetted onto the ridged surface and was mixed with 10 µL of thrombin solution, giving a final fibrinogen concentration of 0.3 mg/mL and final thrombin concentration of 1 U/mL on the slide. The samples were sealed inside a petri dish along with damp paper to ensure a moist atmosphere for the sample and left to polymerize for one hour at 37°C [14]. After polymerization, a pipette was used to gently remove a superficial fibrin mesh, leaving a 2-D fibrin network on the ridged surface. In experiments where beads were used, 30 µL of a 20 nm carboxy-coated microsphere (FluoSpheres™ Carboxylate-Modified Microspheres, Invitrogen by Thermo Fisher Scientific/Life Technologies Corporation, Eugene, OR) (interchangeably referred to as beads) solution, at the specified dilution, was sonicated for 5 minutes and added to the sample. The bead solution was incubated with the fibrin for 1 minute at room temperature and then gently pipetted off. The sample was then washed three times with 30 µL of HBS buffer to ensure removal of any excess bead solution. Following the washes, 20 µL of HBS buffer was placed on top of the sample prior to conducting microscopy imaging. Samples were imaged on a Lieca DMi8 epifluorescent microscope (Leica Microsystems Inc., Buffalo Grove, IL), using a 63x oil-immersion objective. Using light with a wavelength of 480 nm, corresponding to the excitation wavelength of ALEXA-488 labeled fibrinogen, an imaging area was located. Sample areas containing at least 4 individual fibers, each separated by at least 1µm within the camera frame were selected for imaging to maximize the rate of data acquisition. Once an area was found, the microscope settings were changed to a wavelength of 553 nm, the excitation wavelength of the carboxylate-coated polystyrene beads (580 nm) under investigation. We located the fibers using the 480 nm wavelength to minimize fluorescent excitation of the beads while locating the fibers, because the bead excitation spectrum has a minimum ~480 nm, per the manufacturer’s data sheet. This was done because we suspected that the beads may be sensitive to light dosage. While later experiments did not bear this hypothesis out (see results), we maintained this experimental approach. To initiate lysis, plasmin (Enzyme Research Laboratories, South Bend, IN) was added directly to the sample and then time-series acquisition began immediately afterwards. After the addition of plasmin, the attached microscope camera (Leica DFC9000GT SCMOS 4 Megapixel monochrome camera) captured an image every 30 seconds for one hour. The camera shutter was set to remain open during the entire imaging process, unless otherwise stated. When the time-lapse was completed, the file was imported into FIJI image analysis software where the singular fibers were analyzed. In the initial frame, single fibers, all of which presented as tensed structures, were counted and designated for further analysis. Cleaving times were found by analyzing each image series frame-by-frame until finding the frame in which the fiber cleaved. The number of frames prior to cleavage was multiplied by the time interval between frames to find the cleavage time. Additionally, during these intermediate frames the occurrences of bundling were quantified, categorized as two neighboring, largely disconnected fibers, joined to form a singular new fiber. Over the course of imaging, many fibers lost the tension they presented in the initial frames and became loose structures. We identified these fibers that lost their initial tension as “elongated”, and they were counted in the final frame of the timelapse, along with any fibers that remained tensed.