Test cultures of HFLS were grown to 80% confluence on small culture flasks in 4 ml growth medium. Passaged to 96 well plates and normalised for 24 h in standard cell culture medium, the cells were subsequently exposed to test media with human recombinant uPA (5, 20, 40 and 80 μg/ml) and PAI-1 (0.1, 1 and 5 μg/ml) (ProSpec-Tany TechnoGene Ltd., Israel) at both rich (10% FBS) and the hypoxic model (0.1% FBS) culture medium for 24, 48 and 72 h. UPA and PAI-1 concentrations were chosen for bioactive levels both under physiological and pathological processes (74–79), as no data for bioactive concentrations in vivo vs. in vitro were available.
Pai 1
Manufactured by ProSpec
Sourced in Israel
PAI-1 is a laboratory reagent used in various assays and analyses. It functions as a serine protease inhibitor, specifically targeting plasminogen activator inhibitor-1. PAI-1 plays a role in regulating the fibrinolytic system and is commonly used in research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Fibrinogen, by Merck Group (3 mentions)
Thrombin, by Merck Group (3 mentions)
Cacl2, by Fujifilm (3 mentions)
96 well plate, by Corning (3 mentions)
Spectramax paradigm, by Molecular Devices (2 mentions)
Tween 80, by Merck Group (2 mentions)
Tween 80, by MP Biomedicals (1 mentions)
Spectramax paradigm plate reader, by Molecular Devices (1 mentions)
Antithrombin, by Merck Group (1 mentions)
4 protocols using pai 1
1
Modulating uPA and PAI-1 Signaling
2
Turbidity Assay for Fibrin Gel Formation and Lysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The turbidity assay was performed in quadruplicate according to a minor modification of the method of Kim et al.34 (link) at 37 °C in a 96-well plate (Corning, Corning, NY, USA) using a SpectraMax Paradigm (Molecular Devices, Sunnyvale, CA, USA) as a plate reader. Turbidity was monitored once per minute at a 350 nm wavelength and was calculated as the mean value (n = 4) in a volume of 100 μl HBS at pH 7.4.
For fibrin gel formation, 2.0 mg/ml fibrinogen (Sigma, St. Louis, MO, USA); 5 mM CaCl2 (Wako Pure Chemical); 0.01% Tween 80 (MP Biomedical, Santa Ana, CA, USA); and 0.5 NIH unit/ml thrombin (Sigma) were added to 10 μg/ml Fab fragments or 0.3 unit/ml antithrombin (SLS Behring K. K., King of Prussia, PA, USA) as AT III.
For lysis of the fibrin gel, 2.0 mg/ml fibrinogen (Sigma); 5 mM CaCl2 (Wako Pure Chemical); 0.01% Tween 80 (MP Biomedical); 0.5 NIH unit/ml thrombin (Sigma); 0.2 μM PLG (Enzyme Research Laboratories, South Bend, IN, USA); and 0.3 nM tPA (Technoclone, Vienna, Austria) were added to 10 μg/ml Fab fragments or to a mixture of 0.10 μM α2-PI (Hematologic Technologies, Essex Junction, VT, USA) and 2.0 ng/ml PAI-1 (Prospec, East Brunswick, NJ, USA) as a negative control.
For fibrin gel formation, 2.0 mg/ml fibrinogen (Sigma, St. Louis, MO, USA); 5 mM CaCl2 (Wako Pure Chemical); 0.01% Tween 80 (MP Biomedical, Santa Ana, CA, USA); and 0.5 NIH unit/ml thrombin (Sigma) were added to 10 μg/ml Fab fragments or 0.3 unit/ml antithrombin (SLS Behring K. K., King of Prussia, PA, USA) as AT III.
For lysis of the fibrin gel, 2.0 mg/ml fibrinogen (Sigma); 5 mM CaCl2 (Wako Pure Chemical); 0.01% Tween 80 (MP Biomedical); 0.5 NIH unit/ml thrombin (Sigma); 0.2 μM PLG (Enzyme Research Laboratories, South Bend, IN, USA); and 0.3 nM tPA (Technoclone, Vienna, Austria) were added to 10 μg/ml Fab fragments or to a mixture of 0.10 μM α2-PI (Hematologic Technologies, Essex Junction, VT, USA) and 2.0 ng/ml PAI-1 (Prospec, East Brunswick, NJ, USA) as a negative control.
3
Turbidity and Fibrin Gel Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
The turbidity assay was performed as described previously
25 (link)
26 (link)
at 37°C in a 96-well plate (Corning) on a SpectraMax Paradigm plate reader (Molecular Devices, Sunnyvale, California, United States). Assays were performed in quadruplicate. Turbidity was monitored once a minute at a 350 nm wavelength and calculated as the mean value (
n = 4) in a volume of 100 μL HBS (HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]-buffered saline solution) at pH 7.4.
For fibrin gel formation, 2.0 mg/mL fibrinogen (Sigma-Aldrich), 5 mM CaCl
2(Wako Pure Chemical), 0.01% Tween 80 (Sigma-Aldrich), and 0.5 NIH unit/mL thrombin (Sigma-Aldrich) were added to 10 μg/mL 1101 mAb or 3 U/mL anti-thrombin (SLS Behring K) as antithrombin III (AT III).
For the lysis of the fibrin gel, 2.0 mg/mL fibrinogen (Sigma-Aldrich), 5 mM CaCl
2(Wako), 0.01% Tween 80 (Sigma-Aldrich), 0.5 NIH unit/mL thrombin (Sigma-Aldrich), 0.2 μM PLG (Enzyme Research Laboratories), and 0.3 nM tPA (Alteplase, Kyowa Hakko Kirin) were added to 10 μg/mL 1101 mAb or a mixture of 0.10 μM α2-PI (Hematologic Technologies) and 2.0 ng/mL PAI-1 (ProSpec), and used as a negative control.
+ Expand
The turbidity assay was performed as described previously
25 (link)
26 (link)
at 37°C in a 96-well plate (Corning) on a SpectraMax Paradigm plate reader (Molecular Devices, Sunnyvale, California, United States). Assays were performed in quadruplicate. Turbidity was monitored once a minute at a 350 nm wavelength and calculated as the mean value (
n = 4) in a volume of 100 μL HBS (HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]-buffered saline solution) at pH 7.4.
For fibrin gel formation, 2.0 mg/mL fibrinogen (Sigma-Aldrich), 5 mM CaCl
2(Wako Pure Chemical), 0.01% Tween 80 (Sigma-Aldrich), and 0.5 NIH unit/mL thrombin (Sigma-Aldrich) were added to 10 μg/mL 1101 mAb or 3 U/mL anti-thrombin (SLS Behring K) as antithrombin III (AT III).
For the lysis of the fibrin gel, 2.0 mg/mL fibrinogen (Sigma-Aldrich), 5 mM CaCl
2(Wako), 0.01% Tween 80 (Sigma-Aldrich), 0.5 NIH unit/mL thrombin (Sigma-Aldrich), 0.2 μM PLG (Enzyme Research Laboratories), and 0.3 nM tPA (Alteplase, Kyowa Hakko Kirin) were added to 10 μg/mL 1101 mAb or a mixture of 0.10 μM α2-PI (Hematologic Technologies) and 2.0 ng/mL PAI-1 (ProSpec), and used as a negative control.
4
Turbidity Assay and Fibrin Gel Lysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The turbidity assay was performed according to a minor modification of the method of Kim et al. 31 at 37°C in a 96-well plate (Corning) on a SpectraMax Paradigm plate reader (Molecular Devices, Sunnyvale). Measurements were performed in quadruplicate. Turbidity was monitored once a minute at a 350-nm wavelength and calculated as the mean value (n = 4) in a volume of 100 μL HBS (HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]-buffered saline solution) at pH 7.4.
For fibrin gel formation, 2.0 mg/mL fibrinogen (Sigma-Aldrich), 5 mM CaCl 2 (Wako Pure Chemical), 0.01% Tween 80 (Sigma-Aldrich), and 0.5 NIH unit/mL thrombin (Sigma-Aldrich) were added to 10 μg/mL 1101 mAb or 3 U/mL anti-thrombin (SLS Behring K) as AT III (antithrombin III).
For the lysis of the fibrin gel, 2.0 mg/mL fibrinogen (Sigma-Aldrich), 5 mM CaCl 2 (Wako), 0.01% Tween 80 (Sigma-Aldrich), 0.5 NIH unit/mL thrombin (Sigma-Aldrich), 0.2 μM PLG (Enzyme Research Laboratories), and 0.3 nM tPA (Alteplase, Kyowa Hakko Kirin) were added to 10 μg/mL1101 mAb or a mixture of 0.10 μM α2-PI (Hematologic Technologies) and 2.0 ng/mL PAI-1 (ProSpec), and used as a negative control.
For fibrin gel formation, 2.0 mg/mL fibrinogen (Sigma-Aldrich), 5 mM CaCl 2 (Wako Pure Chemical), 0.01% Tween 80 (Sigma-Aldrich), and 0.5 NIH unit/mL thrombin (Sigma-Aldrich) were added to 10 μg/mL 1101 mAb or 3 U/mL anti-thrombin (SLS Behring K) as AT III (antithrombin III).
For the lysis of the fibrin gel, 2.0 mg/mL fibrinogen (Sigma-Aldrich), 5 mM CaCl 2 (Wako), 0.01% Tween 80 (Sigma-Aldrich), 0.5 NIH unit/mL thrombin (Sigma-Aldrich), 0.2 μM PLG (Enzyme Research Laboratories), and 0.3 nM tPA (Alteplase, Kyowa Hakko Kirin) were added to 10 μg/mL1101 mAb or a mixture of 0.10 μM α2-PI (Hematologic Technologies) and 2.0 ng/mL PAI-1 (ProSpec), and used as a negative control.
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