Murine tf standard

Tissue Factor (TF) activity was measured in LSEC and peritoneal macrophages using the method of Cermak et al. (1993) (link). Briefly, at 4 and 24 h after treatment, cells were lysed with 3 freeze-thaw and sonication cycles. TF was determined in the lysates with the coagulant activity assay. Fifty μL of platelet-poor plasma were incubated for 2 min at 37 °C in a borosilicate tube, then 50 μL of the lysate and 50 μL of a 0.025 M CaCl₂ solution were added and the clotting time was measured. A murine TF standard (R&D Systems, USA) was employed to quantify the final concentration of this factor produced by the cells.
Additionally, in peritoneal macrophages TF activity was also measured using the amidolytic assay described by Pereira et al. (2006) (link). Briefly, at 4 and 24 h after treatment, supernatants were discarded and the cells were washed with HEPES saline buffer (50 mM HEPES, 150 mM NaCl, 5 mM CaCl₂, pH 7.4) and incubated for 30 min at room temperature with a mixture containing 5 nM FVIIa and 50 nM FX in HEPES saline buffer. The reaction was stopped with 10 mM EDTA, and TF activity was quantified indirectly with the specific substrate for FXa (S-2765, 2.7 mM). After incubation at 37 °C for 15 and 30 min, the absorbance at 405 nm was measured. A murine TF standard (R&D Systems, USA) was employed to quantify the final concentration of this factor.

Cadmium granules, potassium nitrate, bovine thrombin, sodium chloride, calcium chloride, lipopolysaccharide (LPS, from Escherichia coli serotype 026: B6), trichloroacetic acid, trizma base, sulphorhodamine B, 3′diaminobenzidine, HEPES, and other reagents were purchased from Sigma Aldrich, USA. Avidin peroxidase and streptAvidin peroxidase, anti-VCAM-1, anti-ICAM-1, anti-E-Selectin antibodies were obtained from Santa Cruz Biotechnology, USA. 3,3′,5,5′-tetramethylbenzidine (TMB) was purchased from Scyteck, USA. Chromogenic substrates from Chromogenix AB, Italy. Plasmin, uPA and double chain tPA standards (tcu-PA) were obtained from Sekisui, USA. Mouse IL-6 minikit and mouse TNF-α minikit, recombinant mouse TNF-α were purchased from Thermo Scientific, USA. Fetal bovine serum from Gibco, BRL, USA. vWF antibody from DAKO, USA. Murine TF standard were obtained from R&D Systems, USA.