In-vitro thrombolysis assays have often struggled to replicate in-vivo conditions whether in the model thrombus being digested or in the environment in which thrombolysis is occurring. Herein, we explore how coupling the Chandler loop and Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF) is used for high-fidelity, ex-vivo, clot lysis monitoring.
Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test thrombolytic drug efficiency both in vitro and in vivo. There is increasing demand for more physiologically relevant in-vitro clot models for drug development due to the complexity and cost associated with animal models in addition to their often lack of translatability to human physiology. Flow, pressure, and shear rate are important characteristics of the circulatory system, with clots that are formed under flow displaying different morphology and digestion characteristics than statically formed clots. These factors are often unrepresented in conventional in-vitro clot digestion assays, which can have pharmacological implications that impact drug translational success rates.
The Real-Time Fluorometric Flowing Fibrinolysis (RT-FluFF) assay was developed as a high-fidelity thrombolysis testing platform that uses fluorescently tagged clots formed under shear flow, which are then digested using circulating plasma in the presence or absence of fibrinolytic pharmaceutical agents. Modifying the flow rates of both clot formation and clot digestion steps allows the system to imitate arterial, pulmonary, and venous conditions across highly diverse experimental setups. Measurements can be taken continuously using an in-line fluorometer or by taking discrete time points, as well as a conventional end point clot mass measurement. The RT-FluFF assay is a flexible system that allows for the real-time tracking of clot digestion under flow conditions that more accurately represent in-vivo physiological conditions while retaining the control and reproducibility of an in-vitro testing system.
Diseases fundamentally stemming from thrombo-embolic etiologies present a major source of morbidity and mortality in present-day society. Manifestations of thrombo-embolic pathogenesis include, but are not limited to, myocardial infarctions, ischemic strokes, deep venous thromboses, and pulmonary emboli1. A tremendous amount of ongoing research, spanning multiple disciplines, revolves around the development of safe and effective methods for dealing with pathogenic thrombosis. Variations in arterial and venous manifestations of thrombosis and varying anatomic locations have resulted in the development of different treatment approaches. However, acute treatment generally relies on the use of pharmacologic thrombolysis via plasminogen activators with the potential for mechanical thrombectomy under certain clinical circumstances2.
The development of novel pharmacologic treatment strategies fundamentally relies on both in-vivo animal models and in-vitro digestion models for preclinical testing3,4. In-vivo models naturally benefit from their ability to capture the complex interaction of various physiologic parameters on treatment efficacy that include clearance of pharmaceutical agents as well as cellular interactions with drugs. However, this same complexity often makes such models quite costly and introduces additional issues when attempting to isolate underlying pharmaco-dynamics/kinetics in animals that significantly differ from human physiology. The development of in-vitro models has helped by facilitating a distilled testing setting in which drug development and screening can be performed but often lacks the fidelity necessary to recapitulate the disease state being studied.
Commonly found in-vitro protocols for testing novel thrombolytics rely on the utilization of clots formed and lysed under static conditions whereby the residual clot mass serves as the primary endpoint5,6. Unfortunately, such techniques fail to account for the mechanical aspects of clot lysis such as turbulent flow and trans-thrombus pressure drops that can significantly alter the pharmacodynamics of test drugs. Additionally, clots formed under static conditions contain microarchitecture that differs from physiologic clots. The presence of shear during clot formation has reproducibly been shown to impact the resulting clot characteristics such as platelet activation and fibrin-crosslinking. Clots being produced under shear flow exhibit complex heterogeneity from tip-to-tail that is absent in statically formed clots7,8. Such departures from physiologic clot architecture may impact important drug development characterization that includes drug penetration within a thrombus and subsequent lysis efficiency9.
To address some of these limitations associated with the use of static clotting/clot-lysis models, the adoption of the Chandler loop for both clot formation and clot lysis in the presence of shear has seen a resurgence10. Although such systems allow for a better representation of flow dynamics and generate clots with more physiologically relevant architecture compared to relatively static assays, their simplified flow conditions still represent a deviation from physiologic conditions. Lastly, microfluidic approaches have also been undertaken due to their ease of imaging and uniform flow patterns; however, they remain a significant removal from the physiologic conditions expected within the larger vessels primarily affected in most clinically relevant thrombo-embolic disorders11,12.
With the above discussion in mind, we developed a high-fidelity, in-vitro thrombolysis model for preclinical thrombolytic drug screening. The model aims at addressing some of the current pitfalls detailed above in the realm of novel thrombolytic therapy screening and was validated for reproducibility and sensitivity at varying concentrations of tissue plasminogen activator (tPA). The system described herein offers physiological shear flows utilizing a peristaltic pump, a pressure dampener, a heated reservoir, two pressure sensors, an in-line fluorometer, and a fluorescently labeled Chandler loop shear-formed clot analog to facilitate real-time tracking of fibrinolysis13. Taken together, the overall system is called the Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF Assay)14 and this manuscript will discuss the intricacies of successfully setting up and running assays in this high-fidelity in-vitro thrombolysis model.
All methods mentioned below are in accordance with institutional review board (IRB) protocols and the institutional human research ethics committee. All healthy volunteers provided written and informed consent prior to blood donation. Of note, all materials referenced within the protocol can be found in the Table of Materials. While human WB and plasma are discussed throughout this protocol, the use of research animal blood and factor-depleted blood products can be purchased and substituted.
1. Whole blood collection
2. Clot formation
3. RT-FluFF instrument setup
4. Loading the clot into the flow loop
5. Cleaning the system
Chandler loop clot formation
In forming clots, we generally aimed for quadruplicates to ensure that if any clot outliers (based on gross morphology and mass) existed, we still had the ability to run triplicate thrombolysis assays. Assuming optimal loading conditions, clots should all be quite uniform in length (~3.3 cm), weight (~100 mg), and appearance as is represented in Figure 3. When employing FITC-Fg, we also aimed to examine clots under UV light to ensure relatively uniform dispersion of fluorescence as opposed to irregular areas of hyper-fluorescent labeling within the clot. Varying the degree of FITC-Fg within the whole blood should not alter clot weights or appearances within our explored concentrations; however, micro clot architecture was impacted at the highest FITC-Fg concentrations (1:5 FITC-Fg to unmodified Fg). Prior work done by Zeng et al. captures the wide range of clot phenotypes expected in the Chandler loop depending on the shear level chosen16.
At times, aberrant clotting can occur. The most common clot formation issues fall into one of two categories: (i) premature clotting and (ii) impaired clotting. In premature clotting, the entire volume of blood will solidify within the Chandler loop tubing. This will become apparent when the drum rotation is turned on as the blood will not "flow" but rather stick to the walls of the tubing and rotate in unison. In such circumstances, these clots are not usable and must be discarded. To avoid this, ensure that once clotting is activated with the addition of calcium chloride the loop is immediately closed and placed on the rotating drum for clotting to ensue. The second circumstance, or impaired clotting, occurs when samples have not been adequately mixed or if the blood has started to clot before the rotation is started but not quite as significant as in circumstance (i). Clots with the above issues will tend to appear stringy and flimsy when handled and often have significantly elevated clot masses. Phenotypically, they will approximate clots seen at very low venous shears as this essentially represents clotting in the setting of stasis. Irregular clots should be expected on occasion and discarded.
RT-FluFF fluorometer calibration
An important feature of the RT-FluFF system is the fluorometer, or spectrophotometer, used to track thrombolysis over the elapsed clot digestion period. Prior to any experiments being run, ensuring the proper functioning of the fluorometer, or spectrophotometer, is critical. For our purposes, since we designed our fluorometer specifically for the RT-FluFF apparatus, we needed to ensure it correlated well with the spectrophotometer in the presence of known amounts of FITC-Fg dilutions in a static solution (Figure 4A). Once we were able to confidently determine the linear range of our fluorometer, we next aimed to see how the fluorometer would behave in the presence of flow. We verified functionality by incrementally injecting fluorescein into the flowing solution to monitor a stepwise increase in fluorescence to better understand fluorometer reading reproducibility and determine how rapidly fluorescence equilibrated within the flowing system (Figure 4B). Follow-up experiments were performed by steadily injecting both fluorescein and FITC-Fg into the solution in a continuous manner to better mimic the continuous fluorescent release expected from the fibrinolysis experiments (Figure 4C). The differences in slope depicted between FITC-Fg and FITC are thought to arise from the effects of quenching that occurs when FITC is conjugated in proximity to itself, such as when many FITC molecules are conjugated to a single protein (fibrinogen in this case). Naturally, the stepwise increases and slopes will differ not only based on the concentrations of FITC/FITC-Fg utilized but also the number of FITC molecules per fibrinogen. By extension, it becomes pivotal to control for the degree of FITC to fibrinogen conjugation when performing experiments utilizing the RT-FluFF if the hope is to compare data across numerous clot batches. Through extensive experimentation, we have found that ~14 FITC conjugations per fibrinogen provide for good fluorescent signal to track clot digestion while remaining stable in solution (limited premature aggregation) and with minimal impact on the resulting clot microstructure. It is important to note that anytime pure, non-clotted, FITC-Fg is run through the RT-FluFF apparatus, the exposure of FITC-Fg to high shear may promote its aggregation. This phenomenon is most often observed at the junctions of tubing connections and within the pump head tubing and over time can impact available in-solution fluorescence.
When collecting samples from the flow loop at discrete time points to read fluorescence on a spectrophotometer many of these considerations associated with fluorometer calibration are not necessary. However, it is still necessary to explore the dynamic range of the specific spectrophotometer being utilized to ensure FITC-Fg labeling, the amount of FITC-Fg in the Chandler loop formed clot, and the size of the clot are all optimized in your system. It is recommended that samples are either read immediately following isolation from the system or placed in a 96-well plate for multiplex reads following the experiment's completion. If necessary, samples can be acidified directly following collection to eliminate further enzymatic activity prior to being analyzed.
RT-FluFF clot lysis
To validate the RT-FluFF system utilizing clots formed under arterial shears in the Chandler loop, we employed human plasma as the mobile phase in the apparatus with tPA (Alteplase) as a fibrinolytic agent. Concentrations of tPA explored ranged from 0 to 1,000 ng/mL. To mimic human pulmonary flow conditions, the temperature of the plasma reservoir was maintained at 37 °C and physically raised to 8 cm above the clot level to give an average flow pressure of 12 mmHg with the pump rate adjusted to generate an ~500 s-1 shear flow in the absence of clot. Pulsatile flow dampeners were added to the system to ensure minimal variation between peak and trough pressures from the peristaltic pump to achieve a nearly constant shear rate (Figure 4D). In the absence of a pressure dampener, the output pressure of the peristaltic pump fluctuates significantly due to the nature of how it produces flow by ejecting small packets of liquid through the system as the pump head rotates. Clot lysis was observed over the course of 60 minutes. As expected, rising concentrations of tPA made for increased rates of clot lysis (RFU/minute) and heightened mass loss compared to conditions with less or no tPA. At a tPA concentration of 1,000 ng/mL, ~85% clot lysis was achieved over a 60 min digestion period. Examining gross images of clots undergoing lysis one can appreciate that lysis will primarily occur in the tail regions of the clot before impacting the more densely structured head as seen both by physical degradation of clot architecture and loss of surface fluorescence. Expected stretching of clots will also occur based on loss of mechanical integrity over time in a shear rate-dependent manner.
Although significant differences in clot lysis rates can be appreciated, grossly and based on fluorescence release, there is no perfectly linear correlation between fluorescence release and the amount of fibrinolytic present. This is best appreciated in Figure 5. Fibrinolysis in WB-based clots naturally leads to the release of not only FITC-Fg fragments but also red blood cells (RBCs) that are entrapped within the fibrin networks during clot formation. Release of these RBCs into circulation within the RT-FluFF apparatus will begin to noticeably tinge the circulating plasma red as a result. This colorimetric change in the circulating plasma can negatively impact the fluorescent emission from circulating FITC-Fg being read by the fluorometer sensor. As fibrinolytic concentrations reach the upper limits, the degree of FITC-Fg release into solution overpowers the RBC effects on the fluorescence emissions.
Figure 1: Schematic representation of the Chandler Loop setup. Blood-filled tubes loaded on the Chandler loop drum are submerged in a 37 °C water bath throughout the clot formation process with the lights dimmed. The drum is rotated at a constant rotational rate via a DC motor, drive shaft, and control board. This figure has been modified from Zeng et al14. Please click here to view a larger version of this figure.
Figure 2: Schematic representation of the RT-FluFF system. Important components of the system are identified on the image. A single large-volume dampener is pictured in the image to minimize pulsations in the flow associated with the use of a peristaltic pump. In the absence of an in-line fluorometer, two additional options for periodic sampling include: 1) sampling directly from the reservoir; or 2) incorporating an in-line sampling port to extract the mobile phase from. This figure has been modified from Zeng et al14. Abbreviation: RT-FluFF = Real-Time Fluorometric Flowing Fibrinolysis Assay. Please click here to view a larger version of this figure.
Figure 3: Gross Chandler loop FITC-Fg-tagged WB clot characteristics, including mass and physical appearance under ambient and UV light. (A) Consistent clot formation under diverse clotting conditions is achievable through fine-tuning of the Chandler loop clot formation protocol. It is important to note that clot masses and appearances will vary significantly depending on the tubing diameter, rotation speed, and length of clot formation time. Clots pictured were formed at 37 °C for 1 h at a shear rate of 506 s-1. Scale bars = 20 mm. (B) Masses of clots formed in the Chandler loop utilizing respective ratios of FITC-Fg. The data represents the mean ± standard deviation for greater than or equal to triplicate data points. This figure has been modified from Zeng et al14. Abbreviations: FITC-Fg = fluorescein isothiocyanate-tagged fibrinogen; WB = whole blood. Please click here to view a larger version of this figure.
Figure 4: Fluorometer characterization. (A) RT-FluFF in-line fluorometer comparison against a spectrophotometer in the context of known amounts of FITC-Fg dilutions. (B) Stepwise introduction of fluorescein into the RT-FluFF system to determine fluorometer reproducibility and fluorescence equilibration time in flowing human plasma. (C) Continuous infusions comparing fluorescein and FITC-Fg in the RT-FluFF platform. The differences in slope likely stem from fluorescence quenching in the FITC-Fg group. (D) Outlet pressure sensor (post clot) waveforms associated with the use of different-sized dampeners under the same volumetric flow rate conditions. This figure has been modified from Zeng et al14. Abbreviations: FITC-Fg = fluorescein isothiocyanate-tagged fibrinogen; RT-FluFF = Real-Time Fluorometric Flowing Fibrinolysis Assay. Please click here to view a larger version of this figure.
Figure 5: Thrombolysis in the RT-FluFF platform. (A) Representative slopes of fluorescence rise over the course of 60 min of clot digestion in the presence of circulating tPA. Note the initial thrombolysis lag time as tPA activation of plasminogen and clot digestion is not instantaneous. (B) Percent clot mass lost at various concentrations of tPa and varying thrombolysis modalities that include: the RT-FluFF system, Chandler loop clot digestion under constant shear, and static (no shear) clot digestion. The data represents the mean ± standard deviation for greater than or equal to triplicate data points with double asterisks denoting a p-value < 0.01 and triple asterisks signifying a p-value < 0.001. (C) Gross characteristics of thrombolysis at 200 ng/mL of tPA. This figure has been modified from Zeng et al.14. Abbreviations: RT-FluFF = Real-Time Fluorometric Flowing Fibrinolysis Assay; tPA = tissue plasminogen activator. Please click here to view a larger version of this figure.
Clot formation and labeling
The Chandler loop has been demonstrated to provide an easy and effective means of reproducibly generating clots that mimic in-vivo thrombi16. Fine-tuning parameters such as tubing size, rotational speeds, drum diameter, and clotting time allow for the rapid generation of clots under differing shear conditions that can capture architectural features appreciated in a range of thrombi mimicking both arterial and venous sources. The additional flexibility of being able to introduce markers such as FITC-Fg expands the potential uses of these thrombi as we have demonstrated in the RT-FluFF system. As rudimentary as the underlying principles of the Chandler loop are, there remains a steep initial learning curve associated with consistent clot formation while simultaneously handling blood and blood products throughout the clot formation process. This stems from the fact that coagulation of WB is extremely sensitive to handling in the moments preceding and following the initiation of clotting. Of particular importance is the selection of the tubing used during the clot formation step in the Chandler loop as blood surface interactions can impact clot formation progression. Tubing should be of medical or surgical grade (non-pyrogenic and non-hemolytic) exhibiting low bio-reactivity. As discussed in our results, variation in formed clots is expected in the initial learning phases of the technique while developing one's optimal workflow based on experimental scale and unique assay modifications. The eventual achievement of reproducibility is pivotal given the impact of clot architecture on a clot's behavior when undergoing fibrinolysis. Lastly, variation in coagulation, even amongst healthy volunteers, is also to be expected and should be factored in during experimental design.
RT-FluFF assay
Fluorometric/spectrophotometric quantification of fibrinolysis with FITC-Fg as a marker provides numerous benefits over classic techniques that have primarily relied on endpoint clot mass assessment. The ability to monitor, in real time or near-real time, fibrinolysis dynamics in an environment that approximates physiologic flow parameters represents a significant improvement from traditional fibrinolytic drug screening assays. This assay was intentionally designed at the presented scale to more closely resemble clinically relevant clot sizes, flow field, volumetric flow rate, and clot mass burden rather than taking a fully miniaturized microfluidic approach. Scaling down RT-FluFF apparatus would mean that either smaller diameter tubing and/or shorter distances of tubing would have to be utilized. As a result, the system would begin to deviate from the desired flow patterns and physiologic relevance. Similar to the Chandler loop, the RT-FluFF system allows for the control of parameters such as shear rate by modulating flow pressures under constant flow and control over which flowing media is utilized. Additionally, the RT-Fluff system can also accommodate pulsatile flow and unique clot digestion geometries while still allowing for periodic sampling of mobile phase throughout the digestion process which is not possible in a Chandler loop due to its rotating closed digestion setup. The impact of these parameters on clot lysis has been explored in-depth as part of Zeng et al.14.
Taken together, the Chandler loop-formed clots and the RT-FluFF assay tubing can be of the same diameter or varied at both the clot formation and clot digestion steps to achieve differing levels of occlusion, modeling up through nearly full vessel occlusion to mimic thromboembolism. All assays described herein utilized the same tubing diameter in both the Chandler loop and the RT-FluFF system. With increased tubing diameter in the RT-FluFF system, the mobile phase reservoir volume may need to be increased to accommodate the additional volume needed to fill the system. Increasing the reservoir volume may also increase the necessary amount of thrombolytic/drug needed to be added to the system and would also increase the plasma or mobile phase needs. While the use of autologous plasma would be ideal, it would require exceedingly large amounts of blood to be drawn from individual donors to accommodate for the Chandler loop clot formation and the RT-FluFF mobile phase volumes across all assays and replicates needed to complete an entire experiment. For this reason, type-matched pooled plasma acquired either from a blood donation center or purchased from a commercial vendor was commonly employed. While the overall RT-FluFF assay tubing can be shortened to some degree, there are limitations such that all of the components have enough space to remain in the loop to run the digestion experiments. Of particular importance is the long portion of tubing utilized upstream of the clot itself needing to exceed a minimum length, calculated based on tubing diameter and flow rate, such that laminar flow is achieved by the time flow reaches the suspended clot. Regardless of tubing size selection, the same tubing considerations detailed in the Chandler loop clot formation step should also be applied to the RT-FluFF system tubing. It is important to reiterate that the tubing between the pressure sensors, where the Chandler loop formed clot is fixed in place via two inserted needles in an "X" pattern, must be replaced after every assay to prevent leaks from occurring at the needle puncture sites. The use of needles to hold the clot in place during digestion under flow limits clot bunching and provides clot orientation capabilities relative to the flow direction to improve assay-to-assay consistency. While a single representative clot digestion trial using tPA is described herein, the Chandler loop and RT-FluFF assay provide a high degree of customizability to accommodate a wide variety of clot structures, reporter molecules, shear rates, and digestion conditions to test novel fibrinolytics.
Limitations
As with any of the current platforms or methods utilized to screen fibrinolytic drugs, the RT-FluFF assay also suffers from certain weaknesses that must be acknowledged. First, clots formed in the Chandler loop, although exhibiting motifs commonly present in in-vivo clots, are still formed in a different environment than in-vivo clots where pulsatile flow and the interaction with endothelium exists17 Additionally, even though FITC-Fg concentrations were optimized to minimize the effects on clot architecture, the addition of the modified fibrinogen still represents a departure from physiologic conditions. The mobile phase media choice of plasma can impact the use of a fluorometer as there will be a small amount of background signal that is associated with the protein and lipid content of the plasma that may vary from donor to donor. A potentially significant limitation of the RT-FluFF setup is the negative impact of RBCs on fluorometric reads which restricts the use of WB as a mobile phase when utilizing FITC as a fluorescent clot-digesting marker. A feasible method to mitigate this is to introduce a centrifugation step to pellet RBCs prior to reading discrete time points on a spectrophotometer or to utilize a different clot digestion reporter tag that is less impacted by the presence of RBCs18. Careful consideration should be taken based on assay design to ensure that important variables are controlled for. For example, if plasma is the mobile phase of choice, then it is best to pool plasma across many donors to minimize donor-to-donor clotting and fibrinolysis variability. Lastly, as with any fluorescently tagged protein, the issue of quenching naturally arises due to the FITC tag being present in relative proximity to one another on the surface of the fibrinogen19.
Conclusions and future directions
Studying a dynamic process such as thrombolysis requires an equally dynamic environment in which physiologic conditions can be replicated and multiple parameters are able to be controlled simultaneously. The high-fidelity RT-FluFF system fills this gap and will serve as an important tool in the future development and screening of novel thrombolytics prior to their translation into animal models. RT-FluFF is a highly versatile platform, with multiple abilities that have not been discussed herein but could certainly be incorporated into protocols depending on the unique needs of pharmaceutical drug design, mode of action, or replication of diverse clotting disease states. The benefits we have been able to appreciate within the RT-FluFF system are as follows: (a) variable tubing geometries and configurations, (b) pressure monitoring in real-time correlating to the degree of lumen occlusion, (c) fluorescence measurements in real-time requiring no user intervention, (d) assay duration and sample frequency flexibility, (e) use of a reservoir to mimic in-vivo conditions, (f) in-vivo like shear flow-patterns, (g) option to include pulsatile flow, (h) significant flexibility in the Chandler loop formation of distinct disease representative clot analogs, (i) the ability to test drug impacts on clot formation or clot digestion, and (j) direct clot substrate imaging to track bulk clot digestion through the clear tubing additionally allows for post digestion, longitudinal video analysis.
The authors have nothing to disclose.
Research reported in this publication was supported by the National Heart, Lung, And Blood Institute of the National Institutes of Health under Award Number R01HL167877. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
30 G Disposable Hypodermic Needles | Exel International | 26439 | Other Consumables |
6 mm HSS Lathe Bar Stock Tool 150 mm Long | uxcell | B07SXGSQ82 | Chandler loop, |
96-Well Clear Flat Bottom UV-Transparent Microplate | Corning | 3635 | Other Consumables, Non-treated acrylic copolymer, non-sterile |
Air-Tite Luer-lock Unsterile 60 mL Syringes | Air-Tite | MLB3 | RT-FluFF Apparatus , dampeners |
Arium Mini Plus Ultrapure Water System | Sartorius | NA | DI water source |
Calcium Chloride | Millipore Sigma | C5670 | Other Consumables |
Disposable BP Transducers | AD Instruments | MLT0670 | RT-FluFF Apparatus |
Drager Siemans HemoMed Pod | Drager | 5588822 | RT-FluFF Apparatus |
Drager Siemans Patient Monitor | Drager | SC 7000 | RT-FluFF Apparatus |
Drum (cylinder, diameter 120 mm, width 85 mm) | Chandler loop, | ||
Face Shield | Moxe | SHIELDS10 | Chandler loop, |
Fibrinogen From Human Plasma, Alexa Fluor 488 Conjugate | Thermo Scientific | F13191 | Other Consumables |
Fitting, Polycarbonate, Four-Way Stopcock, Male Luer Lock, Non-Sterile | Masterflex | 30600-04 | RT-FluFF Apparatus |
Fluorescein (FITC) | Thermo Scientific | 119245000 | Other Consumables |
General-Purpose Water Bath | Thermo Scientific | 2839 | Chandler loop, |
Hotplate 4 × 4 | Fisher Scientific | 1152016H | RT-FluFF Apparatus |
Human Source Plasma Fresh-Frozen | Zen-Bio | SER-SPL | Other Consumables, CPDA-1 anticoagulant |
Human Whole Blood | Zen-Bio | SER-WB-SDS | Other Consumables, CPDA-1 anticoagulant |
L/S Easy-Load II Pump Head for High-Performance Precision Tubing, PPS Housing, SS Rotor | Masterflex | 77200-62 | RT-FluFF Apparatus, Pump Head |
L/S Variable-Speed Digital Drive Pump with Remote I/O, 6 to 600 rpm; 90 to 260 VAC | Masterflex | 7528-10 | RT-FluFF Apparatus, Pump |
Motor Speed Controller | CoCocina | ZK-MG | Chandler loop, |
Nalgene Tubing T-Type Connectors | Thermo Scientific | 6151-0312 | RT-FluFF Apparatus |
Peristaltic pump tubing | Masterflex | 06424-15 | Other Consumables |
Phosphate buffered saline | Millipore Sigma | P3813 | Other Consumables, Powder, pH 7.4, for preparing 1 L solutions |
SpectraMax M5 multi-detection microplate reader system (or other fluorescence detection) | Molecular Devices | M5 | RT-FluFF Apparatus |
Switching Power Supply | SoulBay | UC03U | Chandler loop, |
Thermo Scientific National Target All-Plastic Disposable Syringes 10 mL | Thermo Scientific | S751010 | Other Consumables |
Tissue plasminogen activator, human | Millipore Sigma | T0831 | Other Consumables |
Tubing ID 1/4'', OD 3/8'' | Fisher Scientific | AGL00017 | Other Consumables, cut into 1.5cm sections use to connect tubing to T-type connectors |
Tubing ID 5/32", OD 7/32" | Tygon | ND-100-65, ADF 00009 | Other Consumables |
V3 365 nm Mini – Black Light UV Flashlight | uvBeast | uvB-V3-365-MINI | Chandler loop, used to check completed clots |
ZGA37RG ZYTD520 DC Motor, 12 V, 100 rpm | Pangyoo | ZGA37RG | Chandler loop, |