Thrombin generation was measured by calibrated automated thrombinography (40 (link)), essentially as described before (17 (link)). Briefly, coagulation was initiated in normal pooled plasma with 2 pM TF (Dade Innovin), 30 μM phospholipid vesicles (1,2-dioleoyl-sn-glycero-3-phosphoserine/1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine in 20/60/20 M ratio) and 16 mM CaCl2, in the presence of 40 μg/ml thermostable inhibitor of contact activation and 0 to 20 μM tick protein. Thrombin activity was followed using fluorogenic substrate Z-Gly-Gly-Arg-AMC (I-1140; Bachem), which was added to the plasma together with the CaCl2 solution. Fluorescence was read in a Fluoroskan Ascent reader (Thermo Labsystems), and thrombin generation curves were calculated with the Thrombinoscope software (Thrombinoscope). Lag time and peak height were used as the main read-out parameters. Control experiments indicated that none of the tick proteins affected the slope of the thrombin calibrator used to calibrate the assay.
Fluoroskan ascent reader
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Fluoroskan Ascent reader is a fluorometric and luminometric microplate reader designed for high-sensitivity measurements. It can detect a wide range of fluorescent and luminescent signals, making it suitable for various assay types and applications.
Automatically generated - may contain errors
4 protocols using fluoroskan ascent reader
1
Measuring Thrombin Generation by Calibrated Automated Thrombinography
2
Dual-GLO Luciferase Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two days (48 h) after transfections, cells were washed with sterile 1× PBS and lysed, and the transcriptional activity was quantified using the Dual‐GLO Luciferase Assay System (PROMEGA, Madison, WI, USA) as described in the manufacturer's protocol. The relative luciferase activities were measured with a Fluoroskan Ascent reader (Thermo Fisher Scientific, Waltham, MA, USA). Luciferase activity was normalized to pRL–CMV activity and expressed as fold change relative to the activity of empty vector pGL4.10 (defined as 1). Data are presented as mean ± SEM of three independent experiments in triplicate.
3
Thrombin Generation Assay with UFH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Thrombin generation (TG) was performed in platelet-poor citrated human plasma using the Calibrated Automated Thrombogram method, as previously described [26 (link)]. Briefly, to plasma, phospholipids were added in a 20/60/20 (Phosphatidylserine/Phosphatidylcholine/Phosphatidylethanolamine) molar ratio to the concentration of 4 μM. UFH (Smithfield Bioscience) and M6229 were added at the indicated concentrations. Thrombin generation was triggered by PPP Reagent Int-High (based on ellagic acid, reflecting the intrinsic route of coagulation activation, similar to aPTT) and measured using a low-affinity fluorescent thrombin substrate (Z-Gly-Gly-Arg 7-amino-4-methylcoumarin) (Thrombinoscope, Stago, Parsippany, NJ, USA) in a Fluoroskan Ascent reader (Thermo Fisher, Waltham, MA, USA) with the CAT control and evaluation software (version 5.0.0.742, Thrombinoscope, Stago).
4
Measuring MASP1 Activity on Mannan
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The activity of MBL-bound MASP1 was measured as described (39 (link)). In short, Nunc Maxisorb flat-bottom 96-well plates (Thermo Fisher Scientific) were coated with 1 mg/ml mannan and blocked as described previously. Then 50 μl of normal human serum (Sigma–Aldrich) were diluted 1:1 in 20 mM Hepes, 2 M NaCl, 10 mM CaCl2, pH 7.4 and incubated at 4 °C for 1 h. After incubation, wells were washed two times with 100 μl of 10 mM Hepes, 1 M NaCl, 5 mM CaCl2, 0.05% Tween-20, pH 7.4, and another three times with 100 μl of 10 mM Tris, 140 mM NaCl, 5 mM CaCl2, and 0.05% Tween-20. Finally, 200 μl of 20 mM Hepes, 5 mM CaCl2, 0.1 mM Boc-Val-Pro-Arg-7-amido-4-methylcoumarin, pH 8.5 with or without D70-L104 Salp14 was added, and the fluorescent signal (λex = 390 nm, λem = 460 nm) was detected using a Fluoroskan Ascent reader (Thermo Labsystems)
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!