TF activity was measured in in vitro–generated NET structures, using Tissue Factor Human Chromogenic Activity Assay Kit (ab108906, Abcam) in accordance to the manufacturer’s instructions. DNase I (1 U/mL; EN0525, Thermo Fisher Scientific) or anti-TF polyclonal neutralizing antibody (10 μg/mL; 4501, Sekisui Diagnostics) were used as inhibitors.
Tissue factor human chromogenic activity assay kit
Manufactured by Abcam
Sourced in United Kingdom
The Tissue Factor Human Chromogenic Activity Assay Kit is a laboratory product designed to quantify the activity of Tissue Factor, a key component of the blood coagulation cascade. The kit utilizes a chromogenic substrate to measure the enzymatic activity of Tissue Factor in a sample. The assay provides a quantitative assessment of Tissue Factor levels.
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5 protocols using tissue factor human chromogenic activity assay kit
1
In Vitro NET Tissue Factor Activity
2
Measuring Tissue Factor Activity
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After 4 h of cell stimulation, TF activity was measured in cell supernatant [38 (link)] using Tissue Factor Human Chromogenic Activity Assay Kit (ab108906, Abcam), in accordance to the manufacturer's instructions. In brief, the assay measures the ability of TF/FVIIa to activate factor X to Xa. The change in absorbance of the chromophore is directly proportional to the TF enzymatic activity. For the reliability of our results, TF activity was also evaluated directly in the plasma samples that were then used as stimuli in cell cultures. In this case, plasma samples were diluted in DMEM, at the final concentration of 2%, to resemble culture conditions. These values served as controls of the assay.
3
Tissue Factor Activity Assay in Plasma
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Plasma TF procoagulant activity was measured using the Tissue Factor Human Chromogenic Activity Assay Kit (ABCAM, #ab108906) as previously described (26 (link)). Briefly, 10 µL plasma was added to assay mix comprising of 50 µL assay diluent, 10 µL factor VII and 10 µL factor X and mixed. The mixed sample was incubated at 37°C for 30 minutes and subsequently 20 µL FXa substrate was added to the sample. Absorbance at 405 nm was measured every 5 minutes for 35 minutes with a microplate reader. Standard curves were prepared by serially diluting standard solution (500pM) 1:2 with sample diluent which correlated with TF activity.
4
Tissue Factor and Clotting Time Measurement
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PPL-dependent clotting time (PPL-CT) was measured with STA Procoag PPL (Diagnostica Stago, Asnières, France) on an STA Compact Coagulation Analyzer (Diagnostica Stago).
TF Ag was measured by Imubind antihuman Tissue Factor ELISA (Sekisui Diagnostics, Lexington, Massachusetts, United States) using a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, California, United States) measuring the absorbance at a wavelength of 450 nm. In the following, we refer to this method as TF Ag
ELISA.
A supplemental method for TF Ag measurement, represented by a modified version of EV Array with polyclonal anti-TF antibodies (R&D Systems Inc.) as capturing agents and biotin labeled polyclonal anti-TF antibodies (R&D Systems Inc.) as detecting agents, was applied and designated TF Ag
EV Array.
TF activity was evaluated with Tissue Factor Human Chromogenic Activity Assay Kit (Abcam, Cambridge, UK), and the absorbance at a wavelength of 405 nm was measured with SpectraMax M2 Microplate Reader (Molecular Devices).
Lipid levels were measured as routine analyses at the Department of Clinical Biochemistry, Aalborg University Hospital, using a Cobas 8000 Modular Analyzer (Roche Applied Science, Penzberg, Germany).
TF Ag was measured by Imubind antihuman Tissue Factor ELISA (Sekisui Diagnostics, Lexington, Massachusetts, United States) using a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, California, United States) measuring the absorbance at a wavelength of 450 nm. In the following, we refer to this method as TF Ag
ELISA.
A supplemental method for TF Ag measurement, represented by a modified version of EV Array with polyclonal anti-TF antibodies (R&D Systems Inc.) as capturing agents and biotin labeled polyclonal anti-TF antibodies (R&D Systems Inc.) as detecting agents, was applied and designated TF Ag
EV Array.
TF activity was evaluated with Tissue Factor Human Chromogenic Activity Assay Kit (Abcam, Cambridge, UK), and the absorbance at a wavelength of 405 nm was measured with SpectraMax M2 Microplate Reader (Molecular Devices).
Lipid levels were measured as routine analyses at the Department of Clinical Biochemistry, Aalborg University Hospital, using a Cobas 8000 Modular Analyzer (Roche Applied Science, Penzberg, Germany).
5
Tissue Factor Activity Assay for Circulating Microparticles
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Plasma was collected using EDTA as the anticoagulant. TF activity of circulating MPs was analyzed as previously described using a tissue factor human chromogenic activity assay kit (Abcam)17 (link)). The assay measures the ability of lipoprotein TF/factor VIIa (F VIIa) to activate factor X (FX) to factor Xa. Briefly, the desired volume of assay mix was freshly prepared, including assay diluent 50 µL, FVII 10 µL, and FX 10 µL, and 70 µl of the assay mix was added to each well. Then 10 µL TF standard or sample was added to each well and mixed gently. The mixed reaction system was then incubated at 37°C for 30 minutes in a humid incubator. After that, FXa 20 µL was added to each well and incubated at 37°C for 30 minutes. Finally, the absorbance at 405 nm was assayed on a microplate reader.
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