on field was kept for the entire duration of the experiment. When a fibrin clot was not formed a z-stack of 10 optical sections, collected at 1.11 mm BioPQQ web intervals, at up to 1 frame per 20 s from the bottom to the interior of the dish, was captured every 5 minutes. The calculated optical thickness of each slice was 0.93 mm. Collected images were analyzed using FV10-ASW software, and data were expressed as the percentage of fluorescence-positive platelets. Platelet aggregation studies in vitro Platelet aggregation was measured in samples containing PRP mixed with PPP and 50 ml of 0.9% NaCl by the turbidimetric method of Born employing a lumiaggregometer. The aggregometer was calibrated with a sample containing platelets for zero light transmission and a sample containing 150 ml of PPP and 50 ml of 0.9% NaCl for 100% transmission. Samples were stirred at 1,000 rpm. Platelet aggregation agonists were added, and the changes in relative light transmittance produced an aggregation curve over 10 minutes at Fibrin Scaffold Magnifies Platelet’ PS Exposure Concurrent monitoring of platelet intracellular Ca2+ concentration changes and PS exposure PRP was prepared as described above and concentration of platelets was adjusted to 36105 cells per microliter with autologous PPP. For measurement of platelet i PRP was incubated with 1 mM fluo-4 AM together with 10 mM Fura Red AM for precisely 45 min at room temperature. Subsequently platelet suspension was obtained by supplementation of PPP with PRP containing platelets loaded with fluorescent 23462267 dye. Next, the suspension was adequately replenished with either 0.9% NaCl and ANX. The experiment commenced when CaCl2 and thrombin were added to initiate clot formation. The total volume of each sample was 200 ml. Studies were conducted in 35-mm glass bottom dishes at 37uC using CLSM as described above, though images were taken every 4.4 s. Collected images were analyzed using FV10-ASW and 16569706 AquaCosmos 2.6 software. The fluorescence intensity was normalized to each maximal value; the relative fluorescence change is referred to as the “relative fluorescence intensity”. The fluo-4 to Fura Red relative fluorescence intensities ratios were calculated and then used as an index of the relative change in i. Statistical analysis Results are reported as means 6 standard deviations. Statistical comparisons of samples were conducted using the Student’s t-test. Differences were considered significant at P,0.05. Results Validation of the ability of R-6G labeled platelets to aggregate in response to collagen and ADP using a turbidimetric aggregometer To characterize the platelet population used in our studies, abilities of non-labeled platelets and platelets labeled with R-6G to aggregate in response to either collagen or ADP stimulation were compared. No significant differences were revealed between these samples in three separate experiments. The respective suspensions showed comparable responses to both stimuli. TF and thrombin effects on fibrin network formation and platelet PS exposure in CLSM studies CLSM imaging was employed to analyze platelet PS exposure, and thus platelet procoagulant activity, in a fibrin clot. In order to resemble the physiology of hemostasis at the outset TF was used to initiate the coagulation cascade, imitating thrombin generation invivo and consequent fibrin formation. Sequentially diluted concentrations of TF were used to initiate fibrin clot formation in this system, and finally 8000 times dilute

Correlation between observed lysis and staining with Syto9 or propidium iodide for anidulafungin

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